Cell culture compositions with antioxidants and methods for polypeptide production

ABSTRACT

Cell culture media comprising antioxidants are provided herein as are methods of using the media for cell culturing and polypeptide production from cells. Compositions comprising polypeptides, such as therapeutic polypeptides, produced by the methods herein are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 61/799,602, filed Mar. 15, 2013, the contents ofwhich are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to cell culture media comprisingantioxidants, methods of using the media for cell culture andpolypeptide production as well as compositions and kits comprising thepolypeptides produced by the methods provided herein.

BACKGROUND OF THE INVENTION

Cell culture manufacturing technology is widely used for the productionof protein-based products such as pharmaceutical formulations oftherapeutic proteins. Commercial production of protein-based products,such as an antibody product, requires optimization of cell cultureparameters in order for the cell to produce enough of the proteinproduct to meet manufacturing demands. However, when cell cultureparameters are optimized for improving productivity of the proteinproduct it is also necessary to maintain the desired quality attributesof the product such as the glycosylation profile, aggregate levels,charge heterogeneity, and amino acid sequence integrity (Li et al.,mAbs, 2010, 2(5):466-477). Another quality attribute of concern is thecolor of the protein product. Regulatory requirements regardingacceptable color levels for liquid formulations of therapeutic productsfor human use must be met (United States Pharmacopoeia Inc., 2000, p.1926-1927 and Council of Europe. European Pharmacopoeia, 2008, 7^(th)Ed. p. 22). Thus, producing a protein product that has an acceptablecolor is an important aspect of therapeutic protein production.

Recent trends towards the subcutaneous delivery of therapeutic proteins,such as monoclonal antibodies, has been accompanied by an increase inconcentration of the formulated protein substance, for example atconcentrations about 100 mg/mL or greater (Daugherty et al., Adv DrugDeliver Rev, 2006, 58(5-6):686-706). A correlation between increasedcolor intensity in compositions comprising increasing amounts oftherapeutic protein has been observed and this relationship may be duelow-level protein product variants previously unobservable by standardmethods for monitoring color intensity of the formulated product.

Oxidation is a major chemical degradation pathway for proteinpharmaceuticals. For example, methionine, cysteine, histidine,tryptophan, and tyrosine are amino acid residues that are susceptible tooxidation due to their reactivity with reactive oxygen species (ROS) andthis oxidation is often observed in pharmaceutical protein formulationsduring storage. Although it is known that cell culture conditions canimpact quality attributes of the protein product, such as production ofsufficient amounts for large-scale manufacturing, the impact of theseconditions on the color intensity of the final protein product remainsunclear.

There is a continuing need to provide improved and cost-effectivemethods of producing proteins (e.g., antibodies) having acceptableproduct quality attributes such as color intensity. Cell culture media,whether chemically undefined or chemically defined, having componentsthat consistently deliver protein products at lower color intensitieswhile maintaining a desired protein concentration (e.g., ≥100 mg/mL)would find use in the development of protein products, such asantibodies.

BRIEF SUMMARY OF THE INVENTION

In some aspects, the invention herein provides a method of culturing acell comprising a nucleic acid encoding a polypeptide, wherein themethod comprises the step of contacting the cell with a cell culturemedium comprising hypotaurine or an analog or precursor thereof, whereinthe cell culture medium comprising the hypotaurine or an analog ofprecursor thereof reduces the color intensity of a compositioncomprising the polypeptide produced by the cell as compared to the colorintensity of a composition comprising the polypeptide produced by thecell cultured in a cell culture medium that does not comprise thehypotaurine or an analog or precursor thereof. In some embodiments, thecell culture medium comprising the hypotaurine or an analog or precursorthereof reduces the color intensity of a composition comprising thepolypeptide produced by the cell by at least about 0.1% as compared to acomposition comprising the polypeptide produced by the cell cultured ina cell culture medium that does not comprise the hypotaurine or ananalog or precursor thereof. In some embodiments, the cell culturemedium comprising the hypotaurine or an analog or precursor thereofreduces the color intensity of a composition comprising the polypeptideproduced by the cell by about 5% to about 50% as compared to acomposition comprising the polypeptide produced by the cell cultured ina cell culture medium that does not comprise the hypotaurine or ananalog or precursor thereof. In some of the embodiments herein, the cellculture medium comprises the hypotaurine or an analog or precursorthereof at a concentration of at least about 0.0001 mM. In some of theembodiments herein, the cell culture medium comprises the hypotaurine oran analog or precursor thereof at a concentration from about 0.0001 mMto about 500.0 mM. In some of the embodiments herein, the cell culturemedium comprises the hypotaurine or an analog or precursor thereof at aconcentration from about 1.0 mM to about 40.0 mM. In some of theembodiments herein, the cell culture medium comprises the hypotaurine oran analog or precursor thereof at a concentration from about 1.0 mM toabout 10.0 mM. In some of the embodiments herein, the hypotaurine or ananalog or precursor thereof is selected from the group consisting ofhypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinicacid, and taurine. In some of the embodiments herein, the cell culturemedium comprising the hypotaurine or an analog or precursor thereof is achemically defined cell culture medium. In some of the embodimentsherein, the cell culture medium comprising the hypotaurine or an analogor precursor thereof is a chemically undefined cell culture medium. Insome of the embodiments herein, the cell culture medium comprising thehypotaurine or an analog or precursor thereof is a basal cell culturemedium. In some of the embodiments herein, the cell culture mediumcomprising the hypotaurine or an analog or precursor thereof is a feedcell culture medium. In some of the embodiments herein, the cell iscontacted with the cell culture medium comprising the hypotaurine or ananalog or precursor thereof during the cell's growth phase. In some ofthe embodiments herein, the cell is contacted with the cell culturemedium comprising the hypotaurine or an analog or precursor thereofduring the cell's production phase. In some of the embodiments herein,the hypotaurine or an analog or precursor thereof is added to the cellculture medium on at least one day of a cell culture cycle. In some ofthe embodiments herein, the hypotaurine or an analog or precursorthereof is added to the cell culture medium on day 0 of a 14 day cellculture cycle. In any of the embodiments herein, the hypotaurine or ananalog or precursor thereof can be added to the cell culture medium onany day of a cell culture cycle. In some of the embodiments herein, thecell is a mammalian cell. In some of the embodiments herein, themammalian cell is a Chinese Hamster Ovary (CHO) cell. In some of theembodiments herein, the polypeptide is an antibody or fragment thereof.

In other aspects, the invention herein provides methods of culturing acell comprising a nucleic acid encoding a polypeptide, wherein themethod comprises the step of contacting the cell with a cell culturemedium, wherein the cell culture medium comprises one or more ofcomponents (a)-(g): (a) hypotaurine; (b) s-carboxymethylcysteine; (c)carnosine; (d) anserine; (e) butylated hydroxyanisole; (f) lipoic acid;and (g) quercitrin hydrate, and wherein the cell culture mediumcomprising one or more of components (a)-(g) reduces the color intensityof a composition comprising the polypeptide produced by the cell ascompared to a composition comprising the polypeptide produced by thecell cultured in a cell culture medium that does not comprise the one ormore of components (a)-(g). In some embodiments, the cell culture mediumcomprising one or more of components (a)-(g) reduces the color intensityof a composition comprising the polypeptide produced by the cells by atleast about 0.1% as compared to a composition comprising the polypeptideproduced by the cell cultured in a cell culture medium that does notcomprise the one or more of components (a)-(g). In some embodiments, thecell culture medium comprising one or more of components (a)-(g) reducesthe color intensity of a composition comprising the polypeptide producedby the cells by about 5% to about 50% as compared to a compositioncomprising the polypeptide produced by the cell cultured in a cellculture medium that does not comprise the one or more of components(a)-(g). In some of the embodiments herein, the cell culture mediumcomprising one or more of components (a)-(g) comprises the one or morecomponents (a)-(g) in an amount selected from: (a) hypotaurine at aconcentration from at least about 0.0001 mM; (b) s-carboxymethylcysteineat a concentration from at least about 0.0001 mM; (c) carnosine at aconcentration from at least about 0.0001 mM; (d) anserine at aconcentration from at least about 0.0001 mM; (e) butylatedhydroxyanisole at a concentration from at least about 0.0001 mM; (f)lipoic acid at a concentration from at least about 0.0001 mM; and (g)quercitrin hydrate at a concentration from at least about 0.0001 mM. Ina further embodiment, the cell culture medium comprises hypotaurine at aconcentration from about 2.0 mM to about 50.0 mM. In some of theembodiments herein, the cell culture medium comprisess-carboxymethylcysteine at a concentration from about 8.0 mM to about12.0 mM. In some of the embodiments herein, the cell culture mediumcomprises carnosine at a concentration from about 8.0 mM to about 12.0mM. In some of the embodiments herein, the cell culture medium comprisesanserine at a concentration from about 3.0 mM to about 5.0 mM. In someof the embodiments herein, the cell culture medium comprises butylatedhydroxyanisole at a concentration from about 0.025 mM to about 0.040 mM.In some of the embodiments herein, the cell culture medium compriseslipoic acid at a concentration from about 0.040 mM to about 0.060 mM. Insome of the embodiments herein, the cell culture medium comprisesquercitrin hydrate at a concentration from about 0.010 mM to about 0.020mM. In some of the embodiments herein, the cell culture medium is achemically defined cell culture medium. In some of the embodimentsherein, the cell culture medium is a chemically undefined cell culturemedium. In some of the embodiments herein, the cell culture medium is abasal cell culture medium. In some of the embodiments herein, the cellculture medium is a feed cell culture medium. In some of the embodimentsherein, the cell is contacted with the cell culture medium during thecell's growth phase. In some of the embodiments herein, the cell iscontacted with the cell culture medium during the cell's productionphase. In some of the embodiments herein, the one or more of components(a)-(g) is added to the cell culture medium on at least one day of acell culture cycle. In some of the embodiments herein, the one or moreof components (a)-(g) is added to the cell culture medium on day 0 of a14 day cell culture cycle. In any of the embodiments herein, the one ormore of components (a)-(g) can be added to the cell culture medium onany day of a cell culture cycle. In some of the embodiments herein,wherein the cell is a mammalian cell. In some of the embodiments herein,wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell. Insome of the embodiments herein, wherein the polypeptide is an antibodyor fragment thereof.

In some aspects, the invention herein also provides methods of producinga polypeptide comprising the step of culturing a cell comprising anucleic acid encoding the polypeptide in a cell culture mediumcomprising hypotaurine or an analog or precursor thereof, and whereinthe cell culture medium comprising the hypotaurine or an analog orprecursor thereof reduces the color intensity of a compositioncomprising the polypeptide produced by the cell as compared to the colorintensity of a composition comprising the polypeptide produced by thecell cultured in a cell culture medium that does not comprise thehypotaurine or an analog or precursor thereof. In some embodiments, thecell culture medium comprising the hypotaurine or an analog or precursorthereof reduces the color intensity of a composition comprising thepolypeptide produced by the cell by at least about 0.1% as compared to acomposition comprising the polypeptide produced by the cell cultured ina cell culture medium that does not comprise the hypotaurine or ananalog or precursor thereof. In some embodiments, the cell culturemedium comprising the hypotaurine or an analog or precursor thereofreduces the color intensity of a composition comprising the polypeptideproduced by the cell by about 5% to about 50% as compared to acomposition comprising the polypeptide produced by the cell cultured ina cell culture medium that does not comprise the hypotaurine or ananalog or precursor thereof. In some of the embodiments herein, the cellculture medium comprises the hypotaurine or an analog or precursorthereof at a concentration from at least about 0.0001 mM. In some of theembodiments herein, the cell culture medium comprising comprises thehypotaurine or an analog or precursor thereof at a concentration fromabout 0.0001 mM to about 500.0 mM. In some of the embodiments herein,the cell culture medium comprises the hypotaurine or an analog orprecursor thereof at a concentration from about 1.0 mM to about 40.0 mM.In some of the embodiments herein, the cell culture medium comprises thehypotaurine or an analog or precursor thereof at a concentration fromabout 1.0 mM to about 10.0 mM. In some of the embodiments herein, thehypotaurine or an analog or precursor thereof is selected from the groupconsisting of hypotaurine, s-carboxymethylcysteine, cysteamine,cysteinesulphinic acid, and taurine. In some of the embodiments herein,the cell culture medium is a chemically defined cell culture medium. Insome of the embodiments herein, the cell culture medium is a chemicallyundefined cell culture medium. In some of the embodiments herein, thecell culture medium is a basal cell culture medium. In some of theembodiments herein, the cell culture medium is a feed cell culturemedium. In some of the embodiments herein, the hypotaurine or an analogor precursor thereof is added to the cell culture medium on at least oneday of a cell culture cycle. In some of the embodiments herein, thehypotaurine or an analog or precursor thereof is added to the cellculture medium on day 0 of a 14 day cell culture cycle. In any of theembodiments herein, the hypotaurine or an analog or precursor thereofcan be added to the cell culture medium on any day of a cell culturecycle. In some of the embodiments herein, the cell is a mammalian cell.In some embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO)cell. In some of the embodiments herein, the polypeptide is an antibody.In some embodiments, the antibody is an IgG1 antibody. In someembodiments, the antibody is secreted into the cell culture mediumcomprising the hypotaurine or an analog or precursor thereof. In someembodiments, the method further comprises the step of recovering thepolypeptide from the cell culture medium comprising the hypotaurine oran analog or precursor thereof. In some embodiments, the compositioncomprising the recovered polypeptide is a liquid composition or anon-liquid composition. In some embodiments, the composition comprisingthe recovered polypeptide appears as a colorless or slightly coloredliquid.

In some aspects, the invention provides a method of producing apolypeptide comprising the step of culturing a cell comprising a nucleicacid encoding the polypeptide in a cell culture medium, wherein the cellculture medium comprises one or more of components (a)-(g): (a)hypotaurine; (b) s-carboxymethylcysteine; (c) carnosine; (d) anserine;(e) butylated hydroxyanisole; (f) lipoic acid; and (g) quercitrinhydrate, and wherein the cell culture medium comprising one or more ofcomponents (a)-(g) reduces the color intensity of a compositioncomprising the polypeptide produced by the cell as compared to acomposition comprising the polypeptide produced by the cell cultured ina cell culture medium that does not comprise one or more of components(a)-(g). In some embodiments, the cell culture medium comprising one ormore of components (a)-(g) reduces the color intensity of a compositioncomprising the polypeptide produced by the cells by at least about 0.1%as compared to a composition comprising the polypeptide produced by thecell cultured in a cell culture medium that does not comprise the one ormore of components (a)-(g). In some embodiments, the cell culture mediumcomprising one or more of components (a)-(g) reduces the color intensityof a composition comprising the polypeptide produced by the cells byabout 5% to about 50% as compared to a composition comprising thepolypeptide produced by the cell cultured in a cell culture medium thatdoes not comprise the one or more of components (a)-(g). In some of theembodiments herein, the cell culture medium comprises the one or morecomponents (a)-(g) in an amount selected from: (a) hypotaurine at aconcentration from at least about 0.0001 mM; (b) s-carboxymethylcysteineat a concentration from at least about 0.0001 mM; (c) carnosine at aconcentration from at least about 0.0001 mM; (d) anserine at aconcentration from at least about 0.0001 mM; (e) butylatedhydroxyanisole at a concentration from at least about 0.0001 mM; (f)lipoic acid at a concentration from at least about 0.0001 mM; and (g)quercitrin hydrate at a concentration from at least about 0.0001 mM. Insome embodiments, the cell culture medium comprises hypotaurine at aconcentration from about 2.0 mM to about 50.0 mM. In some of theembodiments herein, the cell culture medium comprisess-carboxymethylcysteine at a concentration from about 8.0 mM to about12.0 mM. In some of the embodiments herein, the cell culture mediumcomprises carnosine at a concentration from about 8.0 mM to about 12.0mM. In some of the embodiments herein, the cell culture medium comprisesanserine at a concentration from about 3.0 mM to about 5.0 mM. In someof the embodiments herein, the cell culture medium comprises butylatedhydroxyanisole at a concentration from about 0.025 mM to about 0.040 mM.In some of the embodiments herein, the cell culture medium compriseslipoic acid at a concentration from about 0.040 mM to about 0.060 mM. Insome of the embodiments herein, the cell culture medium comprisesquercitrin hydrate at a concentration from about 0.010 mM to about 0.020mM. In some of the embodiments herein, the cell culture medium is achemically defined cell culture medium. In some of the embodimentsherein, the cell culture medium is a chemically undefined cell culturemedium. In some of the embodiments herein, the cell culture medium is abasal cell culture medium. In some of the embodiments herein, the cellculture medium is a feed cell culture medium. In some of the embodimentsherein, the cell is contacted with the cell culture medium during thecell's growth phase. In some of the embodiments herein, the cell iscontacted with the cell culture medium during the cell's productionphase. In some of the embodiments herein, the one or more of components(a)-(g) is added to the cell culture medium on at least one day of acell culture cycle. In some of the embodiments herein, the one or moreof components (a)-(g) is added to the cell culture medium on day 0 of a14 day cell culture cycle. In any of the embodiments herein, the one ormore of components (a)-(g) can be added to the cell culture medium onany day of a cell culture cycle. In some of the embodiments herein, thecell is a mammalian cell. In some embodiments, the mammalian cell is aChinese Hamster Ovary (CHO) cell. In some of the embodiments herein, thepolypeptide is an antibody or fragment thereof. In some embodiments, theantibody is an IgG1 antibody. In some embodiments, the antibody issecreted into the cell culture medium. In some of the embodimentsherein, the method further comprises the step of recovering thepolypeptide from the cell culture medium comprising one or more ofcomponents (a)-(g). In some embodiments, a composition comprising therecovered polypeptide is a liquid composition or a non-liquidcomposition. In some embodiments, the composition comprising therecovered polypeptide appears as a colorless or slightly colored liquid.In some of the embodiments herein, a polypeptide can be produced by theany of the methods described herein.

In some aspects, the invention provides a pharmaceutical compositioncomprising a polypeptide produced by any of the methods described hereinand a pharmaceutically acceptable carrier.

In some aspects, the invention provides a kit for supplementing a cellculture medium with chemically defined constituents, the kit comprisinghypotaurine or an analog or precursor thereof at a concentration of atleast about 0.0001 mM, and wherein the hypotaurine or an analog orprecursor is selected from the group consisting of hypotaurine,s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, andtaurine.

In other aspects, the invention also provides a kit for supplementing acell culture medium with chemically defined constituents, the kitcomprising one or more of: (a) hypotaurine in an amount to provide fromat least about 0.0001 mM hypotaurine in the cell culture medium; (b)s-carboxymethylcysteine in an amount to provide from at least about0.0001 mM s-carboxymethylcysteine in the cell culture medium; (c)carnosine in an amount to provide from at least about 0.0001 mMcarnosine in the cell culture medium; (d) anserine in an amount toprovide from at least about 0.0001 mM anserine in the cell culturemedium; (e) butylated hydroxyanisole in an amount to provide from atleast about 0.0001 mM butylated hydroxyanisole; (f) lipoic acid in anamount to provide from at least about 0.0001 mM lipoic acid in the cellculture medium; and (g) quercitrin hydrate in an amount to provide fromat least about 0.0001 mM quercitrin hydrate in the cell culture medium.

In some aspects, the invention herein provides a cell culture mediumcomprising from at least about 0.0001 mM of hypotaurine or an analog orprecursor thereof selected from the group consisting of hypotaurine,s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, andtaurine.

In other aspects of the invention, provided herein is a cell culturemedium comprising one or more of components (a)-(g): (a) from at leastabout 0.0001 mM hypotaurine; (b) from at least about 0.0001 mMs-carboxymethylcysteine; (c) from at least about 0.0001 mM carnosine;(d) from at least about 0.0001 mM anserine; (e) from at least about0.0001 mM butylated hydroxyanisole; (f) from at least about 0.0001 mMlipoic acid; and (g) from at least about 0.0001 mM quercitrin hydrate.

In some aspects, the invention herein provides a composition comprising(a) a cell comprising a nucleic acid encoding a polypeptide; and (b) anycell culture medium described herein.

In some aspects of the invention, provided herein is a compositioncomprising: (a) a polypeptide; and (b) any cell culture medium describedherein. In some embodiments, the polypeptide is secreted into the cellculture medium by a cell comprising a nucleic acid encoding thepolypeptide.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of representative compounds screened for impact oncolor intensity in a representative cell culture medium containing anantibody. Numerical results were normalized to the positive controlwhere the value for the positive control was set at 0% change in colorintensity. Values higher than 0% indicate increased color intensity.Values lower than 0% indicate reduced color intensity.

FIG. 2 is a subplot of FIG. 1 showing compounds that reduced colorintensity in a representative cell culture medium containing anantibody. Numerical results were normalized to the positive controlwhere the value for the positive control was set at 0% change in colorintensity. Values lower than 0% indicate reduced color intensity.

FIG. 3 is a series of graphs showing that a shaker flask cell culturemodel is comparable to the corresponding larger scale 2 L cell culturemodel. A) Cell growth in culture over the duration of incubation asmeasured by viable cell density (VCC) and expressed as number of cellsper cell culture volume. B) Cell viability in cell culture over theduration of incubation as measured by the number of viable cells as apercentage of the total number of cells. C) Antibody production in cellculture over the duration of incubation as measured by high performanceliquid chromatography and expressed as antibody titer. SF indicatesshaker flask cell culture model. 2 L indicates a larger scale cellculture model.

FIG. 4 is a series of graphs showing that addition of hypotaurine tocell culture media did not compromise cell growth, cell viability, orantibody production. A) Cell growth in culture over the duration ofincubation as measured by VCC and expressed as number of cells per cellculture volume. B) Cell viability in cell culture over the duration ofincubation as measured by the number of viable cells as a percentage ofthe total number of cells. C) Antibody production in cell culture overthe duration of incubation as measured by high performance liquidchromatography and expressed as antibody titer.

FIG. 5 is a graph showing color intensity of antibody compositionsisolated from cell cultures grown in media supplemented withhypotaurine. 100%, 50%, or 25% indicates basal Media 1 supplemented with9.16 mM, 4.58 mM or 2.29 mM hypotaurine, respectively. Filled circlesindicate color intensity values for cell culture experiments. Emptycircles indicate color intensity values for incubation screeningexperiments. Numerical results were normalized to the positive control,where the value for the positive control was set at 0% change in colorintensity. Values lower than 0% indicate reduced color intensity.

FIG. 6 is a graph showing color intensity of antibody compositionsisolated from cell cultures grown in media supplemented withhypotaurine. 3×, 2×, or 1× indicates basal Media 3 supplemented with38.85 mM, 25.9 mM or 12.95 mM hypotaurine, respectively. Filled circlesindicate color intensity values for cell culture experiments. Numericalresults were normalized to the positive control, where the value for thepositive control was set at 0% change in color intensity. Values lowerthan 0% indicate reduced color intensity.

FIG. 7 contains graphs showing that addition of hypotaurine, or carboxymethyl cysteine to media did not compromise cell growth or cellviability. A) Cell growth in culture over the duration of incubation asmeasured by VCC and expressed as number of cells per cell culturevolume. B) Cell viability in culture over duration of incubationexpressed as percent of total culture volume.

FIG. 8 is a graph showing that addition of hypotaurine, or carboxymethyl cysteine to media did not significantly reduce antibodyproduction. Antibody production in cell culture over the duration ofincubation was measured by high performance liquid chromatography andexpressed as antibody titer.

FIG. 9 is a graph showing color intensity of antibody compositionsisolated from cell cultures grown in media supplemented with hypotaurineor carboxy methyl cysteine. A and B) Indicate two different color assaysused to measure color intensity. Numerical results were normalized tothe positive control where the value for the positive control was set at0% change in color intensity. Values lower than 0% indicate reducedcolor intensity.

DETAILED DESCRIPTION I. Definitions

The terms “medium” and “cell culture medium” refer to a nutrient sourceused for growing or maintaining cells. As is understood by a person ofskill in the art, the nutrient source may contain components required bythe cell for growth and/or survival or may contain components that aidin cell growth and/or survival. Vitamins, essential or non-essentialamino acids, and trace elements are examples of medium components.

A “chemically defined cell culture medium” or “CDM” is a medium with aspecified composition that is free of products derived from animal orplant such as for example animal serum and plant peptone. As would beunderstood by a person of skill in the art, a CDM may be used in aprocess of polypeptide production whereby a cell is in contact with, andsecretes a polypeptide into, the CDM. Thus, it is understood that acomposition may contain a CDM and a polypeptide product and that thepresence of the polypeptide product does not render the CDM chemicallyundefined.

A “chemically undefined cell culture medium” refers to a medium whosechemical composition cannot be specified and which may contain one ormore products derived from animal or plant such as for example animalserum and plant peptone. As would be understood by a person of skill inthe art, a chemically undefined cell culture medium may contain aproduct derived from an animal or a plant as a nutrient source.

“Culturing” a cell refers to contacting a cell with a cell culturemedium under conditions suitable to the survival and/or growth and/orproliferation of the cell.

“Batch culture” refers to a culture in which all components for cellculturing (including the cells and all culture nutrients) are suppliedto the culturing vessel at the start of the culturing process.

The phrase “fed batch cell culture,” as used herein refers to a batchculture wherein the cells and culture medium are supplied to theculturing vessel initially, and additional culture nutrients are fed,continuously or in discrete increments, to the culture during theculturing process, with or without periodic cell and/or product harvestbefore termination of culture.

“Perfusion culture” is a culture by which the cells are restrained inthe culture by, e.g., filtration, encapsulation, anchoring tomicrocarriers, etc., and the culture medium is continuously orintermittently introduced and removed from the culturing vessel.

“Culturing vessel” refers to a container used for culturing a cell. Theculturing vessel can be of any size so long as it is useful for theculturing of cells.

As used herein, “hypotaurine analog” refers to a chemical compound thatis structurally similar to hypotaurine, but differs from hypotaurine inchemical composition (e.g., differs by the number, location or chemicalnature of functional groups or substituents on the hypotaurine core).The hypotaurine analog may or may not have different chemical orphysical properties than hypotaurine and may or may not have improvedactivity in cell culture media as compared to hypotaurine, e.g., furtherreducing the color intensity of a polypeptide (e.g., an antibody)produced in the cell culture media as compared to hypotaurine. Forexample, the hypotaurine analog may be more hydrophilic or it may havealtered reactivity as compared to hypotaurine. The hypotaurine analogmay mimic the chemical and/or biologically activity of hypotaurine(i.e., it may have similar or identical activity), or, in some cases,may have increased or decreased activity as compared to hypotaurine.

The term “titer” as used herein refers to the total amount ofrecombinantly expressed polypeptide produced by a cell culture dividedby a given amount of medium volume. Titer is typically expressed inunits of milligrams of polypeptide per milliliter of medium.

A “nucleic acid,” as used interchangeably herein, refer to polymers ofnucleotides of any length, and include DNA and RNA. The nucleotides canbe deoxyribonucleotides, ribonucleotides, modified nucleotides or bases,and/or their analogs, or any substrate that can be incorporated into apolymer by DNA or RNA polymerase, or by a synthetic reaction. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and their analogs. If present, modification to thenucleotide structure may be imparted before or after assembly of thepolymer.

An “isolated nucleic acid” means and encompasses a non-naturallyoccurring, recombinant or a naturally occurring sequence outside of orseparated from its usual context. An isolated nucleic acid molecule isother than in the form or setting in which it is found in nature.Isolated nucleic acid molecules therefore are distinguished from thenucleic acid molecule as it exists in natural cells. However, anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the protein where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

An “isolated” protein (e.g., an isolated antibody) is one which has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with research, diagnostic ortherapeutic uses for the protein, and may include enzymes, hormones, andother proteinaceous or nonproteinaceous solutes. Isolated proteinincludes the protein in situ within recombinant cells since at least onecomponent of the protein's natural environment will not be present.Ordinarily, however, isolated protein will be prepared by at least onepurification step.

A “purified” polypeptide means that the polypeptide has been increasedin purity, such that it exists in a form that is more pure than itexists in its natural environment and/or when initially produced and/orsynthesized and/or amplified under laboratory conditions. Purity is arelative term and does not necessarily mean absolute purity.

“Contaminants” refer to materials that are different from the desiredpolypeptide product. The contaminant includes, without limitation: hostcell materials, such as CHOP; leached Protein A; nucleic acid; avariant, fragment, aggregate or derivative of the desired polypeptide;another polypeptide; endotoxin; viral contaminant; cell culture mediacomponent, etc.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to polymers of amino acids of any length. The polymer may belinear or branched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. Examples ofpolypeptides encompassed within the definition herein include mammalianproteins, such as, e.g., renin; a growth hormone, including human growthhormone and bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-b; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19 and CD20;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as CA125 (ovariancancer antigen) or HER2, HER3 or HER4 receptor; immunoadhesins; andfragments and/or variants of any of the above-listed proteins as well asantibodies, including antibody fragments, binding to a protein,including, for example, any of the above-listed proteins.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity. An antibody can be human,humanized and/or affinity matured.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that can be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey can be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the invention may be made by avariety of techniques, including, for example, the hybridoma method(e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al.,Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567);phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhuet al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004) and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851(1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile.

“Pharmaceutically acceptable” carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed (Remington's PharmaceuticalSciences (20^(th) edition), ed. A. Gennaro, 2000, Lippincott, Williams &Wilkins, Philadelphia, Pa.). Often the physiologically acceptablecarrier is an aqueous pH buffered solution. Examples of physiologicallyacceptable carriers include buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tweed™, polyethylene glycol (PEG), and Pluronics™.

A “sterile” formulation is aseptic or free or essentially free from allliving microorganisms and their spores.

A “colorless or slightly colored” liquid refers to a liquid compositioncomprising a polypeptide that is measured by quantitative and/orqualitative analysis. Qualitative analysis includes visual inspectionsuch as comparison of the composition comprising the polypeptide to areference standard.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a compound”optionally includes a combination of two or more such compounds, and thelike.

It is understood that aspect and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

II. Cell Culture Media

Cell culture media provided herein may find use in methods (e.g., ofculturing cells and producing polypeptides) and in compositions (e.g.,pharmaceutical formulations) as detailed herein. Media components havebeen identified as capable of providing a polypeptide product (e.g., atherapeutic protein) with acceptable quality attributes, such as anacceptable color intensity. One or more of these identified mediacomponents can be used to provide a polypeptide product with anacceptable color intensity. As used herein, “an acceptable colorintensity” of a polypeptide product (e.g., composition comprising thepolypeptide) can refer to the color intensity required for regulatoryapproval of the polypeptide product or the color intensity desired foruse in assessing consistency in lot to lot batches of the polypeptideproduct. In some embodiments, the one or more media component is anantioxidant. In some embodiments, the one or more media component isselected from the group consisting of hypotaurine,s-carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine,lipoic acid, and quercitrin hydrate. In some embodiments, the one ormore media component is hypotaurine or an analog or precursor thereof.In some embodiments, the hypotaurine or an analog or precursor thereofis selected from the group consisting of hypotaurine,s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, andtaurine. In some embodiments, the one or more media component istaurine, lipoic acid reduced, or carvedilol.

Media components may be added to the cell culture media in forms thatare known in the art. For example, hypotaurine may be provided as acompound identified by CAS number 300-84-5, s-carboxymethylcysteine maybe provided as a compound identified by CAS number 638-23-3, anserinemay be provided as a compound identified by CAS number 10030-52-1,butylated hydroxyanisole may be provided as a compound identified by CASnumber 25013-16-5, carnosine may be provided as a compound identified byCAS number 305-84-0, lipoic acid may be provided as a compoundidentified by CAS number 1200-22-2, quercitrin hydrate may be providedas a compound identified by CAS number 522-12-3. As another example,analogs or precursors of hypotaurine may be provided such ass-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and/ortaurine. In some embodiments, s-carboxymethylcysteine is provided as acompound identified by CAS number 638-23-3, cysteamine is provided as acompound identified by CAS number 60-23-1, cysteinesulphinic acid isprovided as a compound identified by CAS number 1115-65-7, and taurineis provided as a compound identified by CAS number 107-35-7. In someembodiments, a compound listed in Table 4 is provided such as lipoicacid reduced identified by CAS number 462-20-4 or carvedilol identifiedby CAS number 72956-09-3. The media components provided herein can beprovided to the cell culture medium as a salt, a hydrate, or a salthydrate or any other form known to one of skill in the art. The mediacomponents can also be provided to cell culture media as a solution, anextract, or in solid form. In some embodiments herein, the cell culturemedium is a chemically defined medium. In other embodiments herein, thecell culture medium is a chemically undefined medium.

In some aspects, the invention herein provides a cell culture mediumcomprising one or more of the following components: (a) hypotaurine; (b)s-carboxymethylcysteine; (c) carnosine; (d) anserine; (e) butylatedhydroxyanisole; (f) lipoic acid; and (g) quercitrin hydrate. In someembodiments, the cell culture medium comprises 2 or 3 or 4 or 5 or 6 oreach of components (a), (b), (c), (d), (e), (f) and (g). It isunderstood that the cell culture medium provided herein may contain anycombination of components (a), (b), (c), (d), (e), (f) and (g) the sameas if each and every combination were specifically and individuallylisted. For example, it is understood that a cell culture mediumcomprising four of components (a), (b), (c), (d), (e), (f) and (g) maycomprise any combination of the components so long as at least four ofthe components are present.

In some aspects, a cell culture medium as provided herein contains oneor more media components selected from the group consisting of (a)hypotaurine; (b) s-carboxymethylcysteine; (c) carnosine; (d) anserine;(e) butylated hydroxyanisole; (f) lipoic acid; and (g) quercitrinhydrate in amounts as described in Table 1. It is understood that amedium may comprise any one or more of the medium components of Table 1(e.g., any one or more of components (a)-(g), such as a mediumcomprising components (a), (b), (c), (d) and (e) or a medium comprisingcomponents (a), (b) and (g) or a medium comprising only one ofcomponents (a)-(g)) in any of the amounts listed in Table 1, the same asif each and every combination of components and amounts werespecifically and individually listed. In one aspect, the cell culturemedium is a chemically defined medium. In another aspect, the cellculture medium is a chemically undefined medium. In some embodiments, acell culture medium comprises one or more of components (a)-(g), wherein(a) is from at least about 0.0001 mM hypotaurine, (b) is from at leastabout 0.0001 mM s-carboxymethylcysteine, (c) is f from at least about0.0001 mM carnosine, (d) is from at least about 0.0001 mM anserine, (e)is from at least about 0.0001 mM butylated hydroxyanisole, (f) is fromat least about 0.0001 mM lipoic acid, and (g) is from at least about0.0001 mM quercitrin hydrate. In some embodiments, a cell culture mediumcomprises one or more of components (a)-(g), wherein (a) is from about2.0 mM to about 50.0 mM hypotaurine, (b) is from about 8.0 mM to about12.0 mM s-carboxymethylcysteine, (c) is from about 8.0 mM to about 12.0mM carnosine, (d) is from about 3.0 mM to about 5.0 mM anserine, (e) isfrom about 0.025 mM to about 0.040 mM butylated hydroxyanisole, (f) isfrom about 0.040 mM to about 0.060 mM lipoic acid, and (g) is from about0.010 mM to about 0.020 mM quercitrin hydrate.

TABLE 1 Exemplary Amounts of Media Components Component Amount ofComponent in Medium (a) Hypotaurine from about 0.0001 mM to about 920mM; from about 0.001 mM to about 920 mM; from about 0.01 mM to about 920mM; from about 0.1 mM to about 920 mM; from about 0.5 mM to about 920mM; from about 0.0001 mM to about 820 mM; from about 0.0001 mM to about720 mM; from about 0.0001 mM to about 620 mM; from about 0.0001 mM toabout 520 mM; from about 0.0001 mM to about 420 mM; from about 0.0001 mMto about 320 mM; from about 0.0001 mM to about 220 mM; from about 0.0001mM to about 120 mM; from about 0.0001 mM to about 20 mM; from about 1.0mM to about 920 mM; from about 10.0 mM to about 920 mM; from about 20.0mM to about 920 mM; from about 40.0 mM to about 920 mM; from about 80.0mM to about 920 mM; from about 160.0 mM to about 920 mM; from about 320mM to about 920 mM; from about 640 mM to about 920 mM; from about 800 mMto about 920 mM; from about 0.75 mM to about 700 mM; from about 1.0 mMto about 500 mM; from about 1.25 mM to about 300 mM; from about 1.5 mMto 100 mM; from about 1.6 mM to about 90 mM; from about 1.7 mM to about80 mM; from about 1.8 mM to about 70 mM; from about 1.8 mM to about 60mM; from about 1.8 mM to about 50 mM; from about 2 mM to about 50 mM;from about 5 mM to about 50 mM; from about 10 mM to about 50 mM; fromabout 15 mM to about 50 mM; from about 20 mM to about 50 mM; from about30 mM to about 50 mM; from about 40 mM to about 50 mM; about any of0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 9.0or 12 or 25 or 38 or 45 or 50 mM; at least about any of 0.0001 or 0.001or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 9.0 or 12 mM and nomore than about 60 or 55 or 50 or 45 or 40 mM. (b) s- from about 0.0001mM to about 120 mM; from about 0.001 mM to carboxymethyl about 120 mM;from about 0.01 mM to about 120 mM; from about cysteine 0.1 mM to about120 mM; from about 0.5 mM to about 120 mM; from about 0.0001 mM to 100mM; from about 0.0001 mM to about 80 mM; from about 0.0001 mM to about60 mM; from about 0.0001 mM to about 40 mM; from about 0.0001 mM toabout 20 mM; from about 0.0001 mM to about 10 mM; from about 0.0001 mMto about 120 mM; from about 10 mM to about 120 mM; from about 20 mM toabout 120 mM; from about 40 mM to about 120 mM; from about 60 mM toabout 120 mM; from about 80 mM to about 120 mM; from about 100 mM toabout 120 mM; from about 1.0 mM to about 100 mM; from about 2.0 mM toabout 75 mM; from about 3.0 mM to about 50 mM; from about 4.0 mM toabout 25 mM; from about 5.0 mM to about 15 mM; from about 6.0 mM toabout 14 mM; from about 7.0 mM to about 13 mM; from 8.0 mM to about 12mM; about any of 0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or4.0 or 5.0 or 10 or 15 or 20 mM; at least about any of 0.0001 or 0.001or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 8.0 or 10 or 12 mMand no more than about 25 or 20 or 15 mM. (c) Carnosine from about0.0001 mM to about 20 mM; from about 0.001 mM to about 20 mM; from about0.01 mM to about 20 mM; from about 0.1 mM to about 20 mM; from about 0.5mM to about 20 mM; from about 0.0001 mM to about 15 mM; from about0.0001 mM to about 10 mM; from about 0.0001 mM to about 5.0 mM; fromabout 1.0 mM to about 20 mM; from about 5.0 mM to about 20 mM; fromabout 10 mM to about 20 mM; from about 15 mM to about 20 mM; from about2.0 mM to about 18 mM; from about 4.0 mM to about 16 mM; from about 6.0mM to about 14 mM; from about 8.0 mM to about 12 mM; about any of 0.0001or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 6.0 or 7.0or 8.0 or 9.0 or 10 or 11 or 12 or 13 or 14 mM; at least about any of0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 6.0or 7.0 or 8.0 or 9.0 or 10 or 11 and no more than 15 or 14 or 13 mM. (d)Anserine from about 0.0001 mM to about 20 mM; from about 0.001 mM toabout 20 mM; from about 0.01 mM to about 20 mM; from about 0.1 mM toabout 20 mM; from about 0.5 mM to about 20 mM; from about 0.0001 mM toabout 15 mM; from about 0.0001 mM to about 10 mM; from about 0.0001 mMto about 5.0 mM; from about 1.0 mM to about 20 mM; from about 5.0 mM toabout 20 mM; from about 10 mM to about 20 mM; from about 15 mM to about20 mM; from about 1.0 mM to about 15 mM; from about 2.0 mM to about 10mM; from about 3.0 mM to about 5.0 mM; from about 3.2 mM to about 5.0mM; about any of 0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or4.0 or 5.0 or 6.0 or 7.0 or 8.0 mM; at least about any of 0.0001 or0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 mM and no morethan 9.0 or 8.0 or 7.0 or 6.0 mM. (e) Butylated from about 0.0001 mM toabout 0.2 mM; from about 0.001 mM to hydroxyanisole about 0.2 mM; fromabout 0.005 mM to about 0.2 mM; from about 0.0001 mM to about 0.15 mM;from about 0.0001 mM to about 0.1 mM; from about 0.0001 mM to about 0.05mM; from about 0.0001 mM to about 0.04 mM; from about 0.01 mM to about0.2 mM; from about 0.05 mM to about 0.2 mM; from about 0.1 mM to about0.2 mM; from about 0.15 mM to about 0.2 mM; from about 0.01 mM to about0.15 mM; from about 0.015 mM to about 0.1 mM; from about 0.02 mM toabout 0.05 mM; from about 0.025 mM to about 0.04 mM; from about 0.03 mMto about 0.04 mM; about any of 0.0001 or 0.001 or 0.01 or 0.015 or 0.02or 0.025 or 0.03 or 0.035 or 0.04 or 0.045 or 0.05 or 0.055 or 0.06 mM;at least about any of 0.0001 or 0.001 or 0.01 or 0.015 or 0.02 or 0.025or 0.03 or 0.035 or 0.04 mM and no more than 0.06 or 0.055 or 0.05 mM.(f) Lipoic acid from about 0.0001 mM to about 1.5 mM; from about 0.001mM to about 1.5 mM; from about 0.01 mM to about 1.5 mM; from about0.0001 mM to about 1.25 mM; from about 0.0001 mM to about 1.0 mM; fromabout 0.0001 mM to about 0.75 mM; from about 0.0001 mM to about 0.5 mM;from about 0.0001 mM to about 0.25 mM; from about 0.05 mM to about 1.5mM; from about 0.1 mM to about 1.5 mM; from about 0.25 mM to about 1.5mM; from about 0.5 mM to about 1.5 mM; from about 0.75 mM to about 1.5mM; from about 1.0 mM to about 1.5 mM; from about 1.25 mM to about 1.5mM; from about 0.02 mM to about 1.25 mM; from about 0.03 mM to about 1.0mM; from about 0.032 mM to about 0.1 mM; from about 0.034 mM to about0.09 mM; from about 0.036 mM to about 0.08 mM; from about 0.038 mM toabout 0.07 mM; from about 0.04 mM to about 0.06 mM; about any of 0.0001or 0.001 or 0.01 or 0.02 or 0.03 or 0.04 or 0.05 or 0.06 or 0.07 or 0.08or 0.09 mM; at least about any of 0.0001 or 0.001 or 0.01 or 0.02 or0.03 or 0.04 or 0.05 mM and no more than 0.09 or 0.08 or 0.07 mM. (g)Quercitrin from about 0.0001 mM to about 0.04 mM; from about 0.001 mM tohydrate about 0.04 mM; from about 0.005 mM to about 0.04 mM; from about0.001 mM to about 0.035 mM; from about 0.001 mM to about 0.03 mM; fromabout 0.001 mM to about 0.025 mM; from about 0.001 mM to about 0.02 mM;from about 0.001 mM to about 0.015 mM; from about 0.001 mM to about 0.01mM; from about 0.01 mM to about 0.04 mM; from about 0.015 mM to about0.04 mM; from about 0.02 mM to about 0.04 mM; from about 0.025 mM toabout 0.04 mM; from about 0.03 mM to about 0.04 mM; from about 0.035 mMto about 0.04 mM; from about 0.0075 mM to about 0.035 mM; from about0.01 mM to about 0.03 mM; from about 0.015 mM to about 0.025 mM; fromabout 0.01 mM to about 0.02 mM; about any of 0.0001 or 0.001 or 0.01 or0.011 or 0.012 or 0.013 or 0.014 or 0.015 or 0.016 mM; at least aboutany of 0.0001 or 0.001 or 0.011 or 0.012 or 0.013 or 0.014 mM and nomore than 0.02 or 0.019 or 0.018 mM.

In some aspects, the invention herein provides a cell culture mediumcomprising hypotaurine or an analog or precursor thereof selected fromthe group consisting of hypotaurine, s-carboxymethylcysteine,cysteamine, cysteinesulphinic acid, and taurine. In some aspects, thecell culture medium comprises one or more of the following components:(a) hypotaurine; (b) s-carboxymethylcysteine; (c) cysteamine; (d)cysteinesulphinic acid; and (e) taurine. In some embodiments, the cellculture medium comprises 2 or 3 or 4 or each of components (a), (b),(c), (d), and (e). It is understood that the cell culture mediumprovided herein may contain any combination of components (a), (b), (c),(d), and (e) the same as if each and every combination were specificallyand individually listed. For example, it is understood that a cellculture medium comprising three of components (a), (b), (c), (d), and(e) may comprise any combination of the components so long as at leastthree of the components are present. Hypotaurine analogs include forexample s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, andtaurine. Examples of hypotaurine precursors are well known to one ofskill in the art and in some aspects a hypotaurine precursor can be ahypotaurine analog.

In some aspects, a cell culture medium as provided herein containshypotaurine or an analog or precursor thereof in amounts as described inTable 2. It is understood that a medium may comprise any one or more ofthe medium components of Table 2 (e.g., any one or more of components(a)-(e), such as a medium comprising components (a), (b), (c), and (d)or a medium comprising components (a), (b) and (c) or a mediumcomprising only one of components (a)-(e)) in any of the amounts listedin Table 2, the same as if each and every combination of components andamounts were specifically and individually listed. In some embodiments,a cell culture medium comprises hypotaurine or an analog or precursorthereof such as hypotaurine, s-carboxymethylcysteine, cysteamine,cysteinesulphinic acid, and/or taurine at a concentration from at leastabout 0.0001 mM. In some embodiments, a cell culture medium compriseshypotaurine or an analog or precursor thereof such as hypotaurine,s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and/ortaurine at a concentration from about 0.5 mM to about 500.0 mM.

TABLE 2 Exemplary Amounts of Media Components Component Amount ofComponent in Medium (a) Hypotaurine from about 0.0001 mM to about 920mM; from about 0.001 mM to about 920 mM; from about 0.01 mM to about 920mM; from about 0.1 mM to about 920 mM; from about 0.5 mM to about 920mM; from about 0.0001 mM to about 820 mM; from about 0.0001 mM to about720 mM; from about 0.0001 mM to about 620 mM; from about 0.0001 mM toabout 520 mM; from about 0.0001 mM to about 420 mM; from about 0.0001 mMto about 320 mM; from about 0.0001 mM to about 220 mM; from about 0.0001mM to about 120 mM; from about 0.0001 mM to about 20 mM; from about 1.0mM to about 920 mM; from about 10.0 mM to about 920 mM; from about 20.0mM to about 920 mM; from about 40.0 mM to about 920 mM; from about 80.0mM to about 920 mM; from about 160.0 mM to about 920 mM; from about 320mM to about 920 mM; from about 640 mM to about 920 mM; from about 800 mMto about 920 mM; from about 0.75 mM to about 700 mM; from about 1.0 mMto about 500 mM; from about 1.25 mM to about 300 mM; from about 1.5 mMto 100 mM; from about 1.6 mM to about 90 mM; from about 1.7 mM to about80 mM; from about 1.8 mM to about 70 mM; from about 1.8 mM to about 60mM; from about 1.8 mM to about 50 mM; from about 2 mM to about 50 mM;from about 5 mM to about 50 mM; from about 10 mM to about 50 mM; fromabout 15 mM to about 50 mM; from about 20 mM to about 50 mM; from about30 mM to about 50 mM; from about 40 mM to about 50 mM; about any of0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 9.0or 12 or 25 or 38 or 45 or 50 mM; at least about any of 0.0001 or 0.001or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 9.0 or 12 mM and nomore than about 60 or 55 or 50 or 45 or 40 mM. (b) s- from about 0.0001mM to about 120 mM; from about 0.001 mM to carboxymethyl about 120 mM;from about 0.01 mM to about 120 mM; from about cysteine 0.1 mM to about120 mM; from about 0.5 mM to about 120 mM; from about 0.0001 mM to 100mM; from about 0.0001 mM to about 80 mM; from about 0.0001 mM to about60 mM; from about 0.0001 mM to about 40 mM; from about 0.0001 mM toabout 20 mM; from about 0.0001 mM to about 10 mM; from about 0.0001 mMto about 120 mM; from about 10 mM to about 120 mM; from about 20 mM toabout 120 mM; from about 40 mM to about 120 mM; from about 60 mM toabout 120 mM; from about 80 mM to about 120 mM; from about 100 mM toabout 120 mM; from about 1.0 mM to about 100 mM; from about 2.0 mM toabout 75 mM; from about 3.0 mM to about 50 mM; from about 4.0 mM toabout 25 mM; from about 5.0 mM to about 15 mM; from about 6.0 mM toabout 14 mM; from about 7.0 mM to about 13 mM; from 8.0 mM to about 12mM; about any of 0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or4.0 or 5.0 or 10 or 15 or 20 mM; at least about any of 0.0001 or 0.001or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 8.0 or 10 or 12 mMand no more than about 25 or 20 or 15 mM. (c) cysteamine from about0.0001 mM to about 300 mM; from about 0.001 mM to about 300 mM; fromabout 0.01 mM to about 300 mM; from about 0.0001 mM to about 250 mM;from about 0.0001 mM to about 200 mM; from about 0.0001 mM to about 150mM; from about 0.0001 mM to about 100 mM; from about 0.0001 mM to about50 mM; from about 0.0001 mM to about 1 mM; from about 1 mM to about 300mM; from about 50 mM to about 300 mM; from about 100 mM to about 300 mM;from about 150 mM to about 300 mM; from about 200 mM to about 300 mM;from about 250 mM to about 300 mM; from about 0.02 mM to about 300 mM;from about 0.03 mM to about 200 mM; from about 0.04 mM to about 100 mM;from about 0.05 mM to about 50 mM; from about 0.02 mM to about 1 mM;from about 0.04 mM to about 0.8 mM; from about 0.06 mM to about 0.6 mM;from about 0.08 mM to about 0.4 mM; from about 0.1 mM to about 0.2 mM;about any of 0.0001 or 0.001 or 0.01 or 0.02 or 0.05 or 0.1 or 0.25 or0.5 or 1 or 5 or 10 or 25 or 50 or 100 or 200 or 300 mM; at least about0.0001 or 0.001 or 0.01 or 0.02 or 0.05 or 0.1 or 0.25 mM and no morethan about 50 or 40 or 30 mM. (d) from about 0.0001 mM to 100 mM; fromabout 0.001 mM to 100 mM; cysteinesulphinic from about 0.01 mM to 100mM; from about 0.1 mM to 100 mM; acid from about 0.0001 mM to about 80mM; from about 0.0001 mM to about 60 mM; from about 0.0001 mM to about40 mM; from about 0.0001 mM to about 20 mM; from about 0.0001 mM toabout 1 mM; from about 1 to 100 mM; from about 20 mM to about 100 mM;from about 40 mM to about 100 mM; from about 60 mM to about 100 mM; fromabout 80 mM to about 100 mM; from about 0.1 mM to about 50 mM; fromabout 0.2 mM to about 10 mM; from about 0.3 mM to about 1 mM; from about0.1 mM to about 1 mM; from about 0.2 mM to about 0.8 mM; from about 0.3mM to about 0.6 mM; about any of 0.0001 or 0.001 or 0.01 or 0.1 or 0.2or 0.3 or 0.4 or 0.5 or 0.6 or 0.7 or 1 or 10 or 25 or 50 or 100 mM; atleast about 0.0001 or 0.001 or 0.01 or 0.1 or 0.1 or 0.2 or 0.3 or 0.4mM and no more than 20 or 10 or 15 mM. (e) taurine from about 0.0001 mMto 500 mM; from about 0.001 mM to 500 mM; from about 0.01 mM to 500 mM;from about 0.1 mM to 500 mM; from about 0.5 mM to 500 mM; from about0.0001 mM to about 450 mM; from about 0.0001 mM to about 400 mM; fromabout 0.0001 mM to about 350 mM; from about 0.0001 mM to about 300 mM;from about 0.0001 mM to 250 mM; from about 0.0001 mM to 200 mM; fromabout 0.0001 mM to 150 mM; from about 0.0001 mM to 100 mM; from about0.0001 mM to about 50 mM; from about 1 mM to about 500 mM; from about 50mM to about 500 mM; from about 100 mM to about 500 mM; from about 150 mMto about 500 mM; from about 200 mM to about 500 mM; from about 250 mM toabout 500 mM; from about 300 mM to about 500 mM; from about 350 mM toabout 500 mM; from about 400 mM to about 500 mM; from about 450 mM toabout 500 mM; from about 1.0 mM to about 400 mM; from about 2.0 mM toabout 300 mM; from about 3.0 mM to about 200 mM; from about 4.0 mM toabout 100 mM; from about 1.0 mM to about 10 mM; about any of 0.0001 or0.001 or 0.01 or 0.1 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 mM; atleast about any of 0.0001 or 0.001 or 0.01 or 0.1 or 1 or 2 or 3 or 4 or5 or 6 and no more than 13 or 12 or 11 mM.

In some aspects, a cell culture medium provided herein comprises lipoicacid reduced at a concentration of about 0.0001 mM to about 0.5 mM. Insome aspects, a cell culture medium provided herein comprises carvedilolat a concentration of about 0.0001 mM to about 1.5 mM.

Individual media components provided herein may be present in amountsthat result in one or more advantageous properties for culturing cellsand/or polypeptide production from cell culture. Advantageous propertiesinclude, but are not limited to, reduced oxidation of polypeptides incell culture and/or reduced color intensity of a composition comprisinga polypeptide produced by a cell cultured in a cell culture mediaprovided herein. Advantageous properties of the cell culture mediaprovided herein also include reduction of color intensity of acomposition comprising a polypeptide produced by a cell cultured in thecell culture media without affecting one or more product attributes suchas the amount of the polypeptide produced by the cells (e.g., antibodytiter), the glycosylation (e.g., N-glycosylation) profile of thepolypeptide, the polypeptide charge heterogeneity in the composition, orthe amino acid sequence integrity of the polypeptide. In someembodiments, a one or more advantageous property for culturing a cell ina cell culture media provided herein is reduced color intensity of acomposition comprising a polypeptide produced by the cell withoutaffecting cell viability, the amount of the polypeptide produced by thecells, the glycosylation (e.g., N-glycosylation) profile of thepolypeptide, the polypeptide charge heterogeneity in the composition,and/or the amino acid sequence integrity of the polypeptide. In someembodiments, a one or more advantageous property for culturing a cell ina cell culture media provided herein is reduced color intensity of acomposition comprising a polypeptide produced by the cell and reducedoxidation of the polypeptide in cell culture. These advantageousproperties are applicable to methods of culturing a cell comprising anucleic acid encoding a polypeptide of interest and methods of producinga polypeptide of interest in cell culture as described herein.

In some aspects, one more media component selected from the groupconsisting of hypotaurine, s-carboxymethylcysteine, anserine, butylatedhydroxyanisole, carnosine, lipoic acid, and quercitrin hydrate isprovided herein in an amount that results in one or more advantageousproperty for culturing cells and/or polypeptide production from cellculture. In some embodiments, an amount of hypotaurine in cell culturemedia that results in one or more advantageous property is from about0.5 mM to about 100 mM, from about 1.6 mM to about 90 mM, from about 1.7mM to about 80 mM, from about 1.8 mM to about 70 mM, from about 1.9 mMto about 60 mM, from about 2.0 mM to about 50 mM, or from about 1.75 mMto about 50 mM. In some embodiments, an amount ofs-carboxymethylcysteine in cell culture media that results in one ormore advantageous property is from about 0.5 mM to about 120 mM, fromabout 5.0 mM to about 15 mM, from about 6.0 mM to about 14 mM, fromabout 7.0 mM to about 13 mM, or from 8.0 mM to about 12 mM. In someembodiments, an amount of anserine in cell culture media that results inone or more advantageous property is from about 0.5 mM to about 20 mM,from about 2.0 mM to about 10 mM, or from about 3.0 mM to about 5.0 mM.In some embodiments, an amount of butylated hydroxyanisole in cellculture media that results in one or more advantageous property is fromabout 0.005 mM to about 0.2 mM, from about 0.02 mM to about 0.05 mM, orfrom about 0.025 mM to about 0.04 mM. In some embodiments, an amount ofcarnosine in cell culture media that results in one or more advantageousproperty is from about 0.5 mM to about 20 mM, from about 6.0 mM to about14 mM, or from about 8.0 mM to about 12 mM. In some embodiments, anamount of lipoic acid in cell culture media that results in one or moreadvantageous property is from about 0.01 mM to about 1.5 mM lipoic acid,from about 0.036 mM to about 0.08 mM, from about 0.038 mM to about 0.07mM or from about 0.04 mM to about 0.06 mM. In some embodiments, anamount of quercitrin hydrate in cell culture media that results in oneor more advantageous property is from about 0.005 mM to about 0.04 mM,from about 0.01 mM to about 0.03 mM, from about 0.015 mM to about 0.025mM or from about 0.01 mM to about 0.02 mM. In some embodiments, anamount of one more media component selected from the group consisting ofhypotaurine, s-carboxymethylcysteine, anserine, butylatedhydroxyanisole, carnosine, lipoic acid, and quercitrin hydrate in cellculture media that results in one or more advantageous property isprovided in Table 1.

In some aspects, one more media component selected from the groupconsisting of hypotaurine, s-carboxymethylcysteine, cysteamine,cysteinesulphinic acid, and taurine is provided herein in an amount thatresults in one or more advantageous property for culturing cells and/orpolypeptide production from cell culture. In some embodiments, an amountof hypotaurine in cell culture media that results in one or moreadvantageous property is from about 0.5 mM to about 100 mM, from about1.6 mM to about 90 mM, from about 1.7 mM to about 80 mM, from about 1.8mM to about 70 mM, from about 1.9 mM to about 60 mM, from about 2.0 mMto about 50 mM, or from about 1.75 mM to about 50 mM. In someembodiments, an amount of s-carboxymethylcysteine in cell culture mediathat results in one or more advantageous property is from about 0.5 mMto about 120 mM, from about 5.0 mM to about 15 mM, from about 6.0 mM toabout 14 mM, from about 7.0 mM to about 13 mM, or from about 8.0 mM toabout 12 mM. In some embodiments, an amount of cysteamine in cellculture media that results in one or more advantageous property is fromabout 0.01 mM to about 300 mM, from about 0.02 mM to about 1 mM, fromabout 0.04 mM to about 0.8 mM, from about 0.06 mM to about 0.6 mM, fromabout 0.08 mM to about 0.4 mM, or from about 0.1 mM to about 0.2 mM. Insome embodiments, an amount of cysteinesulphinic acid in cell culturemedia that results in one or more advantageous property is from about0.1 mM to 100 mM, from about 0.2 mM to about 10 mM, from about 0.3 mM toabout 1 mM, from about 0.1 mM to about 1 mM, from about 0.2 mM to about0.8 mM, or from about 0.3 mM to about 0.6 mM. In some embodiments, anamount of taurine in cell culture media that results in one or moreadvantageous property is from about 0.5 mM to 500 mM, from about 4.0 mMto about 100 mM, or from about 1.0 mM to about 10 mM. In someembodiments, an amount of one more media component selected from thegroup consisting of hypotaurine, s-carboxymethylcysteine, cysteamine,cysteinesulphinic acid, and taurine in cell culture media that resultsin one or more advantageous property is provided in Table 2.

A cell culture medium provided herein, in one aspect, results in one ormore favorable product quality attributes or advantageous property whenused in a method of producing a polypeptide in cell culture as comparedto quality attributes of the polypeptide when produced in a differentmedium. Reactive oxygen species (ROS) formed through the use of certainmedia components may oxidize specific amino acids on the polypeptide andproduce oxidized polypeptide products. The presence of such oxidizedprotein species may also alter the product quality attributes of aprotein product, such as color intensity, which may be particularlysignificant for polypeptide products that are formulated at anyconcentration such as, but not limited to, a concentration of greaterthan any of about 1 mg/mL, about 10 mg/mL, about 25 mg/mL, about 50mg/mL, or about 75 mg/mL up to 100 mg/mL. In some embodiments, thepresence of oxidized protein species may alter the product qualityattributes of a protein product, such as color intensity, which may beparticularly significant for polypeptide products that are formulated atconcentrations of greater than any of about 100 mg/mL, about 125 mg/mL,about 150 mg/mL, about 175 mg/mL, about 200 mg/mL or about 250 mg/mL.The color intensity of a composition comprising a polypeptide producedwith a media detailed herein (including a composition comprising atleast about 1 mg/mL, about 10 mg/mL, about 50 mg/mL, about 100 mg/mL,about 150 mg/mL, 200 mg/mL or about 250 mg/mL of the polypeptide, suchas an antibody) can be assessed using a color assay such as onedescribed herein or in, but not limited to, the United StatesPharmacopoeia color standard and the European Pharmacopoeia colorstandard. See USP-24 Monograph 631 Color and Achromaticity. UnitedStates Pharmacopoeia Inc., 2000, p. 1926-1927 and Council of Europe.European Pharmacopoeia, 2008, 7^(th) Ed. P. 22, which are incorporatedherein by reference in their entirety. In any of the embodiments herein,a cell culture media provided herein can be used for the preparation ofcompositions comprising a polypeptide that have a reduced colorintensity as compared to a reference solution as measured by a colorassay. For example, the color intensity of a composition (e.g.,pharmaceutical formulation) comprising a polypeptide (e.g., atherapeutic polypeptide) produced using a cell culture medium asprovided herein can be reduced by any amount including, but not limitedto, at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or more as compared toa composition comprising the polypeptide produced using a cell culturemedium that does not comprise the one or more of components of Table 1or Table 2.

Commercially available media such as, but not limited to, Ham's F10(Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma),Dulbecco's Modified Eagle's Medium ([DMEM], Sigma), Luria broth (LB),and Terrific broth (TB) that are suitable for culturing cells may besupplemented with any of the media components as detailed herein (e.g.,by use of a kit as provided). In addition, any of the media described inHam and Wallace, Meth. Enz., 58:44 (1979), Barnes and Sato, Anal.Biochem., 102:255 (1980), Vijayasankaran et al., Biomacromolecules.,6:605:611 (2005), Patkar et al., J Biotechnology, 93:217-229 (2002),U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985; or U.S. Pat. No.5,122,469, the disclosures of all of which are incorporated herein byreference in their entirety, may be supplemented with any of the mediacomponents as detailed herein (e.g., by use of a kit as provided).

In some embodiments, a cell culture medium provided herein comprisescystine and is free of cysteine. In some embodiments, a cell culturemedium provided herein comprises ferric citrate and is free of ferroussulfate. In some embodiments herein, a cell culture medium provided isfree from cysteine and ferrous sulfate. In some embodiments, the mediumis free from cysteine and ferrous sulfate and comprises cystine and/orferric citrate. In any of the embodiments herein, the cell culture mediacan be a basal medium or a feed medium. Amino acids, vitamins, traceelements and other media components at one or two times the rangesspecified in European Patent EP 307,247 or U.S. Pat. No. 6,180,401 maybe used, which documents are herein incorporated by reference in theirentireties.

Any media provided herein may also be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), ions (such as sodium, chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. In some aspects, a cell culturemedium provided herein contains proteins derived from a plant or ananimal. In some embodiments, a cell culture provided herein is free ofproteins derived from a plant or an animal. Any other necessarysupplements may also be included at appropriate concentrations thatwould be known to those skilled in the art.

III. Methods and Uses of the Invention

Provided herein are methods of culturing cells in a cell culture mediaprovided herein for the production of polypeptides of interest. In someaspects, a method is provided for culturing a cell comprising a nucleicacid encoding a polypeptide of interest, wherein the method comprisesthe step of contacting the cell with a cell culture medium, wherein thecell culture medium comprises one or more of components selected fromthe group consisting of hypotaurine, s-carboxymethylcysteine, carnosine,anserine, butylated hydroxyanisole, lipoic acid, and quercitrin hydrate.In some embodiments, a method is provided for culturing a cellcomprising a nucleic acid encoding a polypeptide of interest, whereinthe method comprises the step of contacting the cell with a cell culturemedium, wherein the cell culture medium comprises one or more ofcomponents selected from the group consisting of (a) hypotaurine, (b)s-carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylatedhydroxyanisole, (f) lipoic acid; and (g) quercitrin hydrate, and whereinthe cell culture medium comprising one or more of components (a)-(g)reduces the color intensity of a composition comprising the polypeptideproduced by the cell as compared to a composition comprising thepolypeptide produced by the cell cultured in a cell culture medium thatdoes not comprise the one or more of components (a)-(g). In someembodiments, the color intensity of the composition comprising thepolypeptide is reduced by at least about 0.1%. In some embodiments, thecolor intensity of the composition comprising the polypeptide is reducedby at least about 5%. In some embodiments, the color intensity of thecomposition comprising the polypeptide is reduced by about 10% to about30%. In some of the embodiments herein, the cell culture mediumcomprises one or more components in an amount selected from (a)hypotaurine at a concentration from about 2.0 mM to about 50.0 mM, (b)s-carboxymethylcysteine at a concentration from about 8.0 mM to about12.0 mM, (c) carnosine at a concentration from about 8.0 mM to about12.0 mM, (d) anserine at a concentration from about 3.0 mM to about 5.0mM, (e) butylated hydroxyanisole at a concentration from about 0.025 mMto about 0.040 mM, (f) lipoic acid at a concentration from about 0.040mM to about 0.060 mM, and (g) quercitrin hydrate at a concentration fromabout 0.010 mM to about 0.020 mM. In some of the embodiments herein, theone or more components selected from the group consisting of (a)hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine,(e) butylated hydroxyanisole, (f) lipoic acid; and (g) quercitrinhydrate is added to the cell culture medium on day 0 of a 14 day cellculture cycle.

In some other aspects, a method is provided for culturing a cellcomprising a nucleic acid encoding a polypeptide of interest, whereinthe method comprises the step of contacting the cell with a cell culturemedium comprising the hypotaurine or an analog or precursor thereof. Insome embodiments, a method is provided for culturing a cell comprising anucleic acid encoding a polypeptide of interest, wherein the methodcomprises the step of contacting the cell with a cell culture mediumcomprising the hypotaurine or an analog or precursor thereof, andwherein the cell culture medium comprising the hypotaurine or an analogof precursor thereof reduces the color intensity of a compositioncomprising the polypeptide produced by the cell as compared to the colorintensity of a composition comprising the polypeptide produced by thecell cultured in a cell culture medium that does not comprise thehypotaurine or an analog or precursor thereof. In some embodiments, thecolor intensity of the composition comprising the polypeptide is reducedby at least about 0.1%. In some embodiments, the color intensity of thecomposition comprising the polypeptide is reduced by at least about 5%.In some embodiments, the color intensity of the composition comprisingthe polypeptide is reduced by about 10% to about 30%. In someembodiments herein, the cell culture medium comprises the hypotaurine oran analog or precursor thereof, at a concentration from at least about0.0001 mM. In some embodiments herein, the cell culture medium comprisesthe hypotaurine or an analog or precursor thereof, at a concentrationfrom about 0.5 mM to about 500 mM. In some embodiments, the cell culturemedium comprises the hypotaurine or an analog or precursor thereof, at aconcentration from about 1.0 mM to about 40 mM. In some embodimentsherein, the hypotaurine or an analog or precursor thereof is selectedfrom the group consisting of hypotaurine, s-carboxymethylcysteine,cysteamine, cysteinesulphinic acid, and taurine. In some of theembodiments herein, the hypotaurine or an analog or precursor thereof isadded to the cell culture medium on day 0 of a 14 day cell culturecycle. In some embodiments, the hypotaurine or an analog or precursorthereof is not added to the cell culture medium incrementally over thecourse of a cell culture cycle.

Also provided herein are methods of producing a polypeptide of interestcomprising the step of culturing a cell comprising a nucleic acidencoding the polypeptide in a cell culture medium, wherein the cellculture medium comprises one or more of components selected from thegroup consisting of (a) hypotaurine, (b) s-carboxymethylcysteine, (c)carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic acid,and (g) quercitrin hydrate. In some embodiments, provided herein aremethods of producing a polypeptide of interest comprising the step ofculturing a cell comprising a nucleic acid encoding the polypeptide in acell culture medium, wherein the cell culture medium comprises one ormore of components selected from the group consisting of (a)hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine,(e) butylated hydroxyanisole, (f) lipoic acid, and (g) quercitrinhydrate, and wherein the cell culture medium comprising one or more ofcomponents (a)-(g) reduces the color intensity of a compositioncomprising the polypeptide produced by the cell as compared to acomposition comprising the polypeptide produced by the cell cultured ina cell culture medium that does not comprise one or more of components(a)-(g). In some embodiments, the color intensity of the compositioncomprising the polypeptide is reduced by at least about 0.1%. In someembodiments, the color intensity of the composition comprising thepolypeptide is reduced by at least about 5%. In some embodiments, thecolor intensity of the composition comprising the polypeptide is reducedby about 10% to about 30%. In some of the embodiments herein, the cellculture medium comprises one or more components in an amount selectedfrom (a) hypotaurine at a concentration from about 2.0 mM to about 50.0mM, (b) s-carboxymethylcysteine at a concentration from about 8.0 mM toabout 12.0 mM, (c) carnosine at a concentration from about 8.0 mM toabout 12.0 mM, (d) anserine at a concentration from about 3.0 mM toabout 5.0 mM, (e) butylated hydroxyanisole at a concentration from about0.025 mM to about 0.040 mM, (f) lipoic acid at a concentration fromabout 0.040 mM to about 0.060 mM, and (g) quercitrin hydrate at aconcentration from about 0.010 mM to about 0.020 mM. In some of theembodiments herein, the one or more components selected from the groupconsisting of (a) hypotaurine, (b) s-carboxymethylcysteine, (c)carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic acid;and (g) quercitrin hydrate is added to the cell culture medium on day 0of a 14 day cell culture cycle.

In another aspect, provided herein are methods of producing apolypeptide of interest comprising the step of culturing a cellcomprising a nucleic acid encoding the polypeptide in a cell culturemedium. In some embodiments, provided herein are methods of producing apolypeptide of interest comprising the step of culturing a cellcomprising a nucleic acid encoding the polypeptide in a cell culturemedium, wherein the cell culture medium comprises hypotaurine or ananalog or precursor thereof, and wherein the cell culture mediumcomprising the hypotaurine or an analog of precursor thereof, reducesthe color intensity of a composition comprising the polypeptide producedby the cell as compared to the color intensity of a compositioncomprising the polypeptide produced by the cell cultured in a cellculture medium that does not comprise the hypotaurine or an analog orprecursor thereof. In some embodiments, the color intensity of thecomposition comprising the polypeptide is reduced by at least about0.1%. In some embodiments, the color intensity of the compositioncomprising the polypeptide is reduced by at least about 5%. In someembodiments, the color intensity of the composition comprising thepolypeptide is reduced by about 10% to about 30%. In some embodimentsherein, the cell culture medium comprises the hypotaurine or an analogor precursor thereof, at a concentration from at least about 0.0001 mM.In some embodiments herein, the cell culture medium comprises thehypotaurine or an analog or precursor thereof, at a concentration fromabout 0.5 mM to about 500 mM. In some embodiments, the cell culturemedium comprises the hypotaurine or an analog or precursor thereof, at aconcentration from about 1.0 mM to about 40 mM. In some embodimentsherein, the hypotaurine or an analog or precursor thereof is selectedfrom the group consisting of hypotaurine, s-carboxymethylcysteine,cysteamine, cysteinesulphinic acid, and taurine. In some of theembodiments herein, the hypotaurine or an analog or precursor thereof isadded to the cell culture medium on day 0 of a 14 day cell culturecycle. In some embodiments, the hypotaurine or an analog or precursorthereof is not added to the cell culture medium incrementally over thecourse of a cell culture cycle.

In any of the embodiments herein, the cell culture medium used in themethods described herein can be a chemically defined cell culture mediumof a chemically undefined cell culture medium. The cell culture mediumprovided herein can be used a basal cell culture medium or as a feedcell medium. In some embodiments, a cell culture medium provided hereinis used in a method for culturing the cell during the cell's growthphase. In some embodiments, a cell culture medium provided herein isused in a method for culturing the cell during the cell's productionphase. In any of the methods herein the cell may be a mammalian cellsuch as a CHO cell. In some embodiments, the polypeptide of interest isan antibody or fragment thereof.

In further embodiments herein the polypeptide of interest is recovered.A composition comprising the recovered polypeptide can be subjected toat least one purification step before assessment of color intensityusing a quantitative or qualitative color assay as described herein. Insome embodiments, the composition comprising the recovered polypeptideis a liquid composition or a non-liquid composition. In someembodiments, the liquid composition or non-liquid composition comprisinga recovered polypeptide can be assessed for color intensity using acolor assay as described herein or known in the art. For example, anon-liquid composition comprising the recovered polypeptide can be alyophilized composition that is subsequently reconstituted beforemeasurement of color intensity. In some embodiments herein, the colorintensity of a composition comprising the polypeptide produced by thecell cultured in a cell culture medium provided herein is reduced by atleast 0.1% as compared to the color intensity of a compositioncomprising the polypeptide produced by the cell cultured in a cellculture medium that does not comprise a media component as describedherein (e.g., hypotaurine or an analog or precursor thereof). In someembodiments, the color intensity is reduced by at least about 0.1%, byat least about 0.2%, by at least about 0.3%, by at least about 0.4%, byat least about 0.5%, by at least about 0.6%, by at least about 0.7%, byat least about 0.8%, by at least about 0.8%, or by at least about 0.9%to about 1.0%. In some embodiments, the color intensity is reduced by atleast about 1%, by at least about 2%, by at least about 3%, by at leastabout 4%, by at least about 5%, by at least about 10%, by at least about15%, by at least about 20%, by at least about 25%, by at least about30%, by at least about 35%, by at least about 40%, by at least about45%, by at least about 50%, by at least about 60%, by at least about70%, by at least about 80%, or by at least about 90% to about 100%. Insome embodiments, the color intensity is reduced by about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9% to about 1.0%. In some embodiments, the color intensityis reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%,about 33%, about 34%, about 35%, about 45%, about 50%, about 60%, about70%, about 80%, about 90% to about 100%. In some embodiments, the colorintensity is reduced by from about 1% to about 10%, from about 5% toabout 15%, from about 5% to about 20%, from about 5% to about 25%, fromabout 5% to about 30%, from about 5% to about 35%, from about 5% toabout 40%, from about 5% to about 45%, from about 5% to about 50%, fromabout 10% to about 20%, or from about 15% to about 25%. In someembodiments, a composition comprising a recovered polypeptide appears asa colorless or slightly colored liquid or composition. A liquid orcomposition can be determined to be colorless or slightly colored usinga color assay as described herein or a color assay known to one of skillin the art. In a further embodiment, the composition is a pharmaceuticalcomposition that optionally further comprises a pharmaceuticallyacceptable carrier as described herein.

Methods of administering a polypeptide as detailed herein are alsoprovided. For example, a method is provided for administering to anindividual a formulation comprising a polypeptide, wherein theformulation has the polypeptide at a concentration greater than at leastabout 100 mg/mL, at least about 125 mg/mL, or at least about 150 mg/mLand has a color intensity value greater than B3, B4, B5, B6, B7, B8, orB9 as measured by the COC assay. In some aspects, the color intensityvalue as determined by the COC assay can be any one of, but not limitedto, B, BY, Y, GY, or R, wherein higher values indicate a lighter colorintensity. Formulations comprising a polypeptide of interest may besuitable for injection, such as subcutaneous injection into anindividual (e.g., subcutaneous injection into a human). In some aspects,a formulation comprising a polypeptide of interest suitable forinjection (e.g., suitable for subcutaneous injection) is at aconcentration greater than at least 100 mg/mL, at least 125 mg/mL, or atleast 150 mg/mL and has a color intensity value greater than B3, B4, B5,B6, B7, B8, or B9 as measured by the COC assay. In some aspects, thecolor intensity value as determined by the COC assay can be any one of,but not limited to, B, BY, Y, GY, or R, wherein higher values indicate alighter color intensity.

Other methods are provided throughout, such as in the Brief Summary ofthe Invention and elsewhere.

Polypeptide Production

The cell culture media detailed herein can be used in a method ofculturing cells to produce polypeptides, including particularantibodies. The medium may be used in a method of culturing cells,whether by batch culture, fed batch culture or perfusion culture, andcan be used in a method of producing any polypeptide including anyaspects or embodiments of the polypeptide as described herein. Thepolypeptides produced by the compositions (e.g., a cell cultured in acell culturing medium provided herein) and methods detailed herein andpresent in the compositions (e.g., cell culture media comprising theproduced polypeptide) provided herein may be homologous to the hostcell, or preferably, may be exogenous, meaning that they areheterologous, i.e., foreign, to the host cell being utilized, such as ahuman protein produced by a Chinese hamster ovary cell, or a yeastpolypeptide produced by a mammalian cell. In one variation, thepolypeptide is a mammalian polypeptide (such as an antibody) directlysecreted into the medium by the host cell. In another variation, thepolypeptide is released into the medium by lysis of a cell comprising anucleic acid encoding the polypeptide.

Any polypeptide that is expressible in a host cell may be produced inaccordance with the present disclosure and may be present in thecompositions provided. The polypeptide may be expressed from a gene thatis endogenous to the host cell, or from a gene that is introduced intothe host cell through genetic engineering. The polypeptide may be onethat occurs in nature, or may alternatively have a sequence that wasengineered or selected by the hand of man. An engineered polypeptide maybe assembled from other polypeptide segments that individually occur innature, or may include one or more segments that are not naturallyoccurring.

Polypeptides that may desirably be expressed in accordance with thepresent invention will often be selected on the basis of an interestingbiological or chemical activity. For example, the present invention maybe employed to express any pharmaceutically or commercially relevantenzyme, receptor, antibody, hormone, regulatory factor, antigen, bindingagent, etc.

Methods for producing polypeptides, such as antibodies, in cell cultureare well known in the art. Provided herein are non-limiting exemplarymethods for producing an antibody (e.g., full length antibodies,antibody fragments and multispecific antibodies) in cell culture. Themethods herein can be adapted by one of skill in the art for theproduction of other proteins, such as protein-based inhibitors. SeeMolecular Cloning: A Laboratory Manual (Sambrook et al., 4^(th) ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012);Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,2003); Short Protocols in Molecular Biology (Ausubel et al., eds., J.Wiley and Sons, 2002); Current Protocols in Protein Science, (Horswillet al., 2006); Antibodies, A Laboratory Manual (Harlow and Lane, eds.,1988); Culture of Animal Cells: A Manual of Basic Technique andSpecialized Applications (R. I. Freshney, 6^(th) ed., J. Wiley and Sons,2010) for generally well understood and commonly employed techniques andprocedures for the production of proteins (e.g., therapeutic proteins),which are all incorporated herein by reference in their entirety.

(A) Antibody Preparation

The antibody produced in cell culture using a cell culture mediumprovided herein is directed against an antigen of interest. Preferably,the antigen is a biologically important polypeptide and administrationof compositions comprising the antibody to a mammal suffering from adisorder can result in a therapeutic benefit in that mammal.

(i) Antigen Preparation

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(ii) Certain Antibody-Based Methods

Monoclonal antibodies of interest can be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), and furtherdescribed, e.g., in Hongo et al., Hybridoma, 14 (3): 253-260 (1995),Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed. 1988); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), and Ni,Xiandai Mianyixue, 26(4):265-268 (2006) regarding human-humanhybridomas. Additional methods include those described, for example, inU.S. Pat. No. 7,189,826 regarding production of monoclonal human naturalIgM antibodies from hybridoma cell lines. Human hybridoma technology(Trioma technology) is described in Vollmers and Brandlein, Histologyand Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein,Methods and Findings in Experimental and Clinical Pharmacology,27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507.An exemplary protocol for producing monoclonal antibodies using thehybridoma method is described as follows. In one embodiment, a mouse orother appropriate host animal, such as a hamster, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization. Antibodiesare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of a polypeptide of interest or a fragment thereof, andan adjuvant, such as monophosphoryl lipid A (MPL)/trehalosedicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton,Mont.). Serum from immunized animals is assayed for anti-antigenantibodies, and booster immunizations are optionally administered.Lymphocytes from animals producing anti-antigen antibodies are isolated.Alternatively, lymphocytes may be immunized in vitro.

Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986). Myeloma cells may be used that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Preferably, serum-free hybridoma cell culturemethods are used to reduce use of animal-derived serum such as fetalbovine serum, as described, for example, in Even et al., Trends inBiotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cellcultures are described in Franek, Trends in Monoclonal AntibodyResearch, 111-122 (2005). Specifically, standard culture media areenriched with certain amino acids (alanine, serine, asparagine,proline), or with protein hydrolyzate fractions, and apoptosis may besignificantly suppressed by synthetic oligopeptides, constituted ofthree to six amino acid residues. The peptides are present at millimolaror higher concentrations.

Culture medium in which hybridoma cells are growing may be assayed forproduction of monoclonal antibodies. The binding specificity ofmonoclonal antibodies produced by hybridoma cells may be determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).The binding affinity of the monoclonal antibody can be determined, forexample, by Scatchard analysis. See, e.g., Munson et al., Anal.Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.See, e.g., Goding, supra. Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In some embodiments,the hybridoma cells are cultured in a cell culture medium providedherein. In some embodiments, the hybridoma cells are cultured in a cellculture medium comprising one or more media components selected from thegroup consisting of hypotaurine, s-carboxymethylcysteine, anserine,butylated hydroxyanisole, carnosine, lipoic acid, and quercitrinhydrate. In some embodiments, the one or more media component ishypotaurine or an analog or precursor thereof. In some embodiments, thehypotaurine or an analog or precursor thereof is selected from the groupconsisting of hypotaurine, s-carboxymethylcysteine, cysteamine,cysteinesulphinic acid, and taurine.

Antibodies may be produced using recombinant methods. For recombinantproduction of an anti-antigen antibody, nucleic acid encoding theantibody is isolated and inserted into a replicable vector for furthercloning (amplification of the DNA) or for expression. DNA encoding theantibody may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.

(iii) Certain Library Screening Methods

Antibodies can be made by using combinatorial libraries to screen forantibodies with the desired activity or activities. For example, avariety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are described generally inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001). For example, one method ofgenerating antibodies of interest is through the use of a phage antibodylibrary as described in Lee et al., J. Mol. Biol. (2004),340(5):1073-93.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the antibodies of interest can be obtained bydesigning a suitable antigen screening procedure to select for the phageclone of interest followed by construction of a full length antibodyclone using the Fv sequences from the phage clone of interest andsuitable constant region (Fc) sequences described in Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NIHPublication 91-3242, Bethesda Md. (1991), vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-antigen clones is desired, the subject is immunizedwith antigen to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In one embodiment, a human antibodygene fragment library biased in favor of anti-antigen clones is obtainedby generating an anti-antigen antibody response in transgenic micecarrying a functional human immunoglobulin gene array (and lacking afunctional endogenous antibody production system) such that antigenimmunization gives rise to B cells producing human antibodies againstantigen. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-antigen reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing antigen-specific membrane bound antibody, e.g., by cellseparation using antigen affinity chromatography or adsorption of cellsto fluorochrome-labeled antigen followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which antigenis not antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, library diversity is maximized by usingPCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹ M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, antigen can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panning phagedisplay libraries.

The phage library samples are contacted with immobilized antigen underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for antigen.However, random mutation of a selected antibody (e.g. as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting antigen, rare high affinity phage could be competed out. Toretain all higher affinity mutants, phages can be incubated with excessbiotinylated antigen, but with the biotinylated antigen at aconcentration of lower molarity than the target molar affinity constantfor antigen. The high affinity-binding phages can then be captured bystreptavidin-coated paramagnetic beads. Such “equilibrium capture”allows the antibodies to be selected according to their affinities ofbinding, with sensitivity that permits isolation of mutant clones withas little as two-fold higher affinity from a great excess of phages withlower affinity. Conditions used in washing phages bound to a solid phasecan also be manipulated to discriminate on the basis of dissociationkinetics.

Anti-antigen clones may be selected based on activity. In certainembodiments, the invention provides anti-antigen antibodies that bind toliving cells that naturally express antigen or bind to free floatingantigen or antigen attached to other cellular structures. Fv clonescorresponding to such anti-antigen antibodies can be selected by (1)isolating anti-antigen clones from a phage library as described above,and optionally amplifying the isolated population of phage clones bygrowing up the population in a suitable bacterial host; (2) selectingantigen and a second protein against which blocking and non-blockingactivity, respectively, is desired; (3) adsorbing the anti-antigen phageclones to immobilized antigen; (4) using an excess of the second proteinto elute any undesired clones that recognize antigen-bindingdeterminants which overlap or are shared with the binding determinantsof the second protein; and (5) eluting the clones which remain adsorbedfollowing step (4). Optionally, clones with the desiredblocking/non-blocking properties can be further enriched by repeatingthe selection procedures described herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of interest is readily isolated and sequenced using conventionalprocedures (e.g. by using oligonucleotide primers designed tospecifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones can be combined with known DNA sequencesencoding heavy chain and/or light chain constant regions (e.g. theappropriate DNA sequences can be obtained from Kabat et al., supra) toform clones encoding full or partial length heavy and/or light chains.It will be appreciated that constant regions of any isotype can be usedfor this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-antigen antibody derived from a hybridoma can also bemodified, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of homologous murinesequences derived from the hybridoma clone (e.g. as in the method ofMorrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNAencoding a hybridoma- or Fv clone-derived antibody or fragment can befurther modified by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In this manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of the Fv clone or hybridomaclone-derived antibodies of interest.

(iv) Humanized and Human Antibodies

Various methods for humanizing non-human antibodies are known in theart. For example, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one embodiment of the method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human antibodies of interest can be constructed by combining Fv clonevariable domain sequence(s) selected from human-derived phage displaylibraries with known human constant domain sequence(s) as describedabove. Alternatively, human monoclonal antibodies of interest can bemade by the hybridoma method. Human myeloma and mouse-humanheteromyeloma cell lines for the production of human monoclonalantibodies have been described, for example, by Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); andBoerner et al., J. Immunol., 147: 86 (1991).

It is possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);and Duchosal et al. Nature 355:258 (1992).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

(v) Antibody Fragments

Antibody fragments may be generated by traditional means, such asenzymatic digestion, or by recombinant techniques. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors. For areview of certain antibody fragments, see Hudson et al. (2003) Nat. Med.9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two differentepitopes (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is typical to have thefirst heavy-chain constant region (CH1) containing the site necessaryfor light chain binding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. One interface comprises at least a part of the C_(H) 3 domainof an antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al, J. Immunol, 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60(1991).

(vii) Single-Domain Antibodies

In some embodiments, an antibody of interest is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

(viii) Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

(B) Vectors, Host Cells, and Recombinant Methods

Antibodies produced by a cell cultured in a cell culture medium providedherein may also be produced using recombinant methods. For recombinantproduction of an anti-antigen antibody, nucleic acid encoding theantibody is isolated and inserted into a replicable vector for furthercloning (amplification of the DNA) or for expression. DNA encoding theantibody may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.

(i) Signal Sequence Component

An antibody may be produced recombinantly not only directly, but also asa fusion polypeptide with a heterologous polypeptide, which ispreferably a signal sequence or other polypeptide having a specificcleavage site at the N-terminus of the mature protein or polypeptide.The heterologous signal sequence selected preferably is one that isrecognized and processed (e.g., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processa native antibody signal sequence, the signal sequence is substituted bya prokaryotic signal sequence selected, for example, from the group ofthe alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxinII leaders. For yeast secretion the native signal sequence may besubstituted by, e.g., the yeast invertase leader, a factor leader(including Saccharomyces and Kluyveromyces α-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in WO 90/13646. In mammalian cell expression, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, are available.

(ii) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ, plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp 1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(iv) Promoter Component

Expression and cloning vectors generally contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding an antibody. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, β-lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. lichenifonnis (e.g., B. lichenifonnis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragmentscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) that by itself showseffectiveness in tumor cell destruction. Full length antibodies havegreater half-life in circulation. Production in E. coli is faster andmore cost efficient. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et.al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523(Simmons et al.), which describes translation initiation region (TIR)and signal sequences for optimizing expression and secretion. See alsoCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli. After expression, the antibody may beisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g., in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger. For a reviewdiscussing the use of yeasts and filamentous fungi for the production oftherapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414(2004).

Certain fungi and yeast strains may be selected in which glycosylationpathways have been “humanized,” resulting in the production of anantibody with a partially or fully human glycosylation pattern. See,e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describinghumanization of the glycosylation pathway in Pichia pastoris); andGerngross et al., supra.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to theinvention, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also beutilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498,6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technologyfor producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 andSp2/0. For a review of certain mammalian host cell lines suitable forantibody production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 255-268.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in a cell culturemedium provided herein modified as appropriate for inducing promoters,selecting transformants, or amplifying the genes encoding the desiredsequences.

Cell Growth and Polypeptide Production

Generally the cells are combined (contacted) with any of the cellculture media described herein under one or more conditions that promoteany of cell growth, maintenance and/or polypeptide production. Methodsof culturing a cell and producing a polypeptide employ a culturingvessel (bioreactor) to contain the cell and cell culture medium. Theculturing vessel can be composed of any material that is suitable forculturing cells, including glass, plastic or metal. Typically, theculturing vessel will be at least 1 liter and may be 10, 100, 250, 500,1000, 2500, 5000, 8000, 10,000 liters or more. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan. Culturing conditions that may be adjustedduring the culturing process include but are not limited to pH andtemperature.

A cell culture is generally maintained in the initial growth phase underconditions conducive to the survival, growth and viability (maintenance)of the cell culture. The precise conditions will vary depending on thecell type, the organism from which the cell was derived, and the natureand character of the expressed polypeptide.

The temperature of the cell culture in the initial growth phase will beselected based primarily on the range of temperatures at which the cellculture remains viable. For example, during the initial growth phase,CHO cells grow well at 37° C. In general, most mammalian cells grow wellwithin a range of about 25° C. to 42° C. Preferably, mammalian cellsgrow well within the range of about 35° C. to 40° C. Those of ordinaryskill in the art will be able to select appropriate temperature ortemperatures in which to grow cells, depending on the needs of the cellsand the production requirements.

In one embodiment of the present invention, the temperature of theinitial growth phase is maintained at a single, constant temperature. Inanother embodiment, the temperature of the initial growth phase ismaintained within a range of temperatures. For example, the temperaturemay be steadily increased or decreased during the initial growth phase.Alternatively, the temperature may be increased or decreased by discreteamounts at various times during the initial growth phase. One ofordinary skill in the art will be able to determine whether a single ormultiple temperatures should be used, and whether the temperature shouldbe adjusted steadily or by discrete amounts.

The cells may be cultured during the initial growth phase for a greateror lesser amount of time. In one variation, the cells are cultured for aperiod of time sufficient to achieve a viable cell density that is agiven percentage of the maximal viable cell density that the cells wouldeventually reach if allowed to grow undisturbed. For example, the cellsmay be cultured for a period of time sufficient to achieve a desiredviable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 99 percent of maximal viable cell density.

In another embodiment the cells are allowed to grow for a defined periodof time. For example, depending on the starting concentration of thecell culture, the temperature at which the cells are cultured, and theintrinsic growth rate of the cells, the cells may be cultured for 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or moredays. In some cases, the cells may be allowed to grow for a month ormore.

The cell culture may be agitated or shaken during the initial culturephase in order to increase oxygenation and dispersion of nutrients tothe cells. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor during theinitial growth phase, including but not limited to pH, temperature,oxygenation, etc. For example, pH can be controlled by supplying anappropriate amount of acid or base and oxygenation can be controlledwith sparging devices that are well known in the art.

An initial culturing step is a growth phase, wherein batch cell cultureconditions are modified to enhance growth of recombinant cells, toproduce a seed train. The growth phase generally refers to the period ofexponential growth where cells are generally rapidly dividing, e.g.growing. During this phase, cells are cultured for a period of time,usually, but not limited to, 1 to 4 days, e.g. 1, 2, 3, or 4 days, andunder such conditions that cell growth is optimal. The determination ofthe growth cycle for the host cell can be determined for the particularhost cell by methods known to those skilled in the art.

In the growth phase, a basal culture medium provided herein and cellsmay be supplied to the culturing vessel in batch. The culture medium inone aspect contains less than about 5% or less than 1% or less than 0.1%serum and other animal-derived proteins. However, serum andanimal-derived proteins can be used if desired. At a particular point intheir growth, the cells may form an inoculum to inoculate a culturemedium at the start of culturing in the production phase. Alternatively,the production phase may be continuous with the growth phase. The cellgrowth phase is generally followed by a polypeptide production phase.

During the polypeptide production phase, the cell culture may bemaintained under a second set of culture conditions (as compared to thegrowth phase) conducive to the survival and viability of the cellculture and appropriate for expression of the desired polypeptide. Forexample, during the subsequent production phase, CHO cells expressrecombinant polypeptides and proteins well within a range of 25° C. to38° C. Multiple discrete temperature shifts may be employed to increasecell density or viability or to increase expression of the recombinantpolypeptide or protein. In one aspect, a medium as provided hereinreduces the presence of metabolic by-products when used in a method ofincreasing polypeptide production as compared to contaminants obtainedwhen the polypeptide is produced in a different medium. In onevariation, the contaminants are reactive oxygen species. In one aspect,a medium as provided herein reduces color intensity of a polypeptideproduct when used in a method of increasing production of thepolypeptide as compared to color intensity obtained when the polypeptideproduct is produced in a different media. In one variation, a method ofincreasing polypeptide production comprises a temperature shift stepduring the polypeptide production phase. In a further variation, atemperature shift step comprises a shift of the temperature from 31° C.to 38° C., from 32° C. to 38° C., from 33° C. to 38° C., from 34° C. to38° C., from 35° C. to 38° C., from 36° C. to 38° C., from 31° C. to 32°C., from 31° C. to 33° C., from 31° C. to 34° C., from 31° C. to 35° C.,or from 31° C. to 36° C.

The cells may be maintained in the subsequent production phase until adesired cell density or production titer is reached. In one embodiment,the cells are maintained in the subsequent production phase until thetiter to the recombinant polypeptide reaches a maximum. In otherembodiments, the culture may be harvested prior to this point. Forexample, the cells may be maintained for a period of time sufficient toachieve a viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximal viablecell density. In some cases, it may be desirable to allow the viablecell density to reach a maximum, and then allow the viable cell densityto decline to some level before harvesting the culture.

In certain cases, it may be beneficial or necessary to supplement thecell culture during the subsequent production phase with nutrients orother medium components that have been depleted or metabolized by thecells. For example, it might be advantageous to supplement the cellculture with nutrients or other medium components observed to have beendepleted during monitoring of the cell culture. Alternatively oradditionally, it may be beneficial or necessary to supplement the cellculture prior to the subsequent production phase. As non-limitingexamples, it may be beneficial or necessary to supplement the cellculture with hormones and/or other growth factors, particular ions (suchas sodium, chloride, calcium, magnesium, and phosphate), buffers,vitamins, nucleosides or nucleotides, trace elements (inorganiccompounds usually present at very low final concentrations), aminoacids, lipids, or glucose or other energy source.

A component provided herein (e.g., hypotaurine an analog or precursorthereof) can be added to the cell culture medium at any time during thecell culture cycle. For example, hypotaurine may be added at any one ormore of days 0-14 for a 14 day cell culture cycle (e.g., at any one ormore of days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) at anyamount to provide a cell culture medium comprising hypotaurine at aconcentration provided herein (e.g., at least 0.0001 mM). It istherefore appreciated that for a 14 day cell culture cycle, hypotaurinemay be added at any one or more of days 0-14 (e.g., at any one or moreof days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) in anyamount. As used herein “day 0” can refer to a cell culture medium thathas been supplemented with a component provided herein (e.g.,hypotaurine) before the cell culture medium has been applied to the cellculture. It is understood that a cell culture cycle can be any amount ofdays as long as the cells remain viable and/or sufficient levels ofpolypeptide are produced as can be determined by one of skill in theart. For example, a cell culture cycle can be at least 3 days, 4 days, 5days, 6 days, at 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 daysin duration. In some embodiments, a component provided herein (e.g.,hypotaurine or an analog or precursor thereof) is added to the cellculture medium on at least on day of a cell culture cycle.

Polypeptide Purification

The polypeptide of interest preferably is recovered from the culturemedium as a secreted polypeptide, although it also may be recovered fromhost cell lysates when directly expressed without a secretory signal. Inone aspect, the polypeptide produced is an antibody, such as amonoclonal antibody.

The culture medium or lysate may be centrifuged to remove particulatecell debris. The polypeptide thereafter may be purified from contaminantsoluble proteins and polypeptides, with the following procedures beingexemplary of suitable purification procedures: by fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; and protein A Sepharosecolumns to remove contaminants such as IgG. A protease inhibitor such asphenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibitproteolytic degradation during purification. One skilled in the art willappreciate that purification methods suitable for the polypeptide ofinterest may require modification to account for changes in thecharacter of the polypeptide upon expression in recombinant cellculture. Polypeptides can be generally purified using chromatographictechniques (e.g., protein A, affinity chromatography with a low pHelution step and ion exchange chromatography to remove processimpurities). For antibodies, the suitability of protein A as an affinityligand depends on the species and isotype of any immunoglobulin Fcdomain that is present in the antibody. Protein A can be used to purifyantibodies that are based on human γ1, γ2, or γ4 heavy chains (Lindmarket al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended forall mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575(1986)). Purified proteins may concentrated to provide a concentratedprotein drug product as described herein, e.g., one with a proteinconcentration of at least 1 mg/mL or 10 mg/mL or 50 mg/mL or 75 mg/mL or100 mg/mL or 125 mg/mL or 150 mg/mL or a concentration of about 1 mg/mLor 10 mg/mL or 50 mg/mL or 75 mg/mL or 100 mg/mL or 125 mg/mL or 150mg/mL. It is understood that concentrated polypeptide products may beconcentrated up to levels that are permissible under the concentrationconditions, e.g., up to a concentration at which the polypeptide is nolonger soluble in solution. For example, a polypeptide purificationprocess can comprise the steps of harvesting cell culture fluid frompolypeptide-producing cells and purifying the polypeptide throughprotein A affinity chromatography with further purification throughanion and cation exchange chromatography, filtration for removal ofvirus, and a final ultrafiltration and diafiltration step for finalformulation and concentration of the polypeptide. Non-limiting examplesof methods for producing and purifying polypeptides for drugformulations are described in Kelley, B. MAbs., 2009, 1(5):443-452,which is incorporated herein in its entirety by reference.

Polypeptide Color Assessment

The polypeptides produced by the methods detailed herein and present inthe compositions provided may be assessed for color at any step of theprotein purification process. A method for assessing color may involveharvesting the cell culture fluid from cells cultured in the mediadetailed herein, purifying the polypeptide from cell culture fluid toobtain a composition (e.g., a solution) comprising the polypeptide andassessing the solution comprising the polypeptide for color. In onevariation, a composition comprising the polypeptide is assessed forcolor after purification with Protein A affinity chromatography. In afurther variation, a composition comprising the polypeptide is assessedfor color after purification by ion exchange chromatography. In anothervariation, a composition comprising the polypeptide is assessed forcolor after purification by high performance liquid chromatography. Inyet another variation, a composition comprising the polypeptide isassessed for color after purification by hydrophobic interactionchromatography. In still another variation, a composition comprising thepolypeptide is assessed for color after purification by size exclusionchromatography. In one variation, a composition comprising thepolypeptide is assessed for color after purification by filtrationincluding microfiltration or ultrafiltration. In one variation, thecomposition comprising the polypeptide is concentrated prior toassessing for color (e.g., the composition may comprise at least 1mg/mL, 10 mg/mL, 50 mg/mL, 75 mg/mL, 100 mg/mL, 125 mg/mL or 150 mg/mLpolypeptide, such as an antibody). The composition comprising thepolypeptide can be concentrated by centrifugation, filter devices,semi-permeable membranes, dialysis, precipitation, ion exchangechromatography, affinity chromatography, high performance liquidchromatography, or hydrophobic interaction chromatography. In onevariation, the polypeptide can be concentrated by lyophilization andresuspended prior to assessment for color. The composition comprisingthe polypeptide may be assessed for color after purification with one ormore of the techniques detailed herein. Color assessment of thecomposition comprising the polypeptide after the composition hasundergone one or more freeze thaw cycle(s) is contemplated herein.Methods for color assessment of cell culture fluid containing thepolypeptide prior to purification or concentration of the polypeptide isfurther contemplated herein.

The polypeptides produced by the methods detailed herein with the mediadescribed herein (or present in the compositions provided) may beassessed for color by use of one or more visual color standards. Methodsfor color assessment of composition comprising the polypeptide includeuse of an international or national color standard such as, but notlimited to, the United States Pharmacopoeia color standard and theEuropean Pharmacopoeia color standard. See USP-24 Monograph 631 Colorand Achromaticity. United States Pharmacopoeia Inc., 2000, p. 1926-1927and Council of Europe. European Pharmacopoeia, 2008, 7^(th) Ed. P. 22,which are incorporated herein by reference in their entirety. Forexample, the Color, Opalescence and Coloration (COC) assay may be usedto assess color of a solution containing the polypeptide. In onevariation, identical tube of colorless, transparent, neutral glass of 12mm external diameter are used to compare 2.0 mL of the compositioncomprising the polypeptide with 2.0 mL of water or of the solvent or ofthe reference solution prescribed in the monograph. The colors arecompared in diffused daylight and viewed horizontally against a whitebackground for color determination, measurement, or assessment. Inanother variation, identical tubes of colorless, transparent, neutralglass with a flat base and an internal diameter of 15 mm to 25 mm areused to compare the composition comprising the polypeptide with water orthe solvent or the reference solution prescribed in the monograph, thedepth of the layer being 40 mm. The colors are compared in diffuseddaylight and viewed vertically against a white background for colordetermination, measurement, or assessment. In one variation, colordetermination, measurement or assessment can be done by human visualinspection. In another variation, color determination, measurement, orassessment can be done by using an automated process. For example, thetubes can be loaded in a machine that images the tubes for processing ofthe images with an algorithm to determine, measure, or assess the color.It is understood that the reference standards for the COC assay can beany one of, but not limited to, brown (B), brownish-yellow (BY), yellow(Y), greenish-yellow (GY), or red (R). Compositions comprising thepolypeptide that are compared to the brown reference standard can begiven a brown reference standard value of B1 (darkest), B2, B3, B4, B5,B6, B7, B8, or B9 (lightest). Compositions comprising the polypeptidethat are compared to the brownish-yellow reference standard can be givena brownish-yellow reference standard value of BY1 (darkest), BY2, BY3,BY4, BY5, BY6, or BY7 (lightest). Compositions comprising thepolypeptide that are compared to the yellow reference standard can begiven a yellow reference standard value of Y1 (darkest), Y2, Y3, Y4, Y5,Y6, or Y7 (lightest). Compositions comprising the polypeptide that arecompared to the greenish-yellow reference standard can be given agreenish-yellow reference standard value of GY1 (darkest), GY2, GY3,GY4, GY5, GY6, or GY7 (lightest). Compositions comprising thepolypeptide that are compared to the red reference standard can be givena red reference standard value of R1 (darkest), R2, R3, R4, R5, R6, orR7 (lightest). In one aspect, an acceptable color is any color exceptthat which measures darkest on a scale provided herein (e.g., except R1for a red reference standard value). In one variation, the color of thecomposition comprising the polypeptide produced by cells cultured in themedia detailed herein has a reference standard value as described inTable 3. As is described herein, it is understood that in one aspect themedia that may be used in the methods and compositions herein result ina polypeptide composition (which in one variation is a compositioncomprising at least 100 mg/mL or 125 mg/mL or 150 mg/ml polypeptide)having a reference standard color value selected from the groupconsisting of B3, B4, B5, B6, B7, B8, B9, BY3, BY4, BY5, BY6, BY7, Y3,Y4, Y5, Y6, Y7, GY3, GY4, GY5, GY6, GY7, R3, R4, R5, R6 and R7. In oneaspect, the media that may be used in the methods and compositionsherein result in a polypeptide composition (which in one variation is acomposition comprising at least 100 mg/mL or 125 mg/mL or 150 mg/mlpolypeptide) having a reference standard color value of greater than anyone of B4, B5, B6, B7, B8, BY4, BY5, BY6, Y4, Y5, Y6, GY4, GY5, GY6,GY7, R3, R4, R5 and R6. As would be understood to the skilled artisan,descriptions of reference standard color values are applicable to, andmay further modify descriptions of, any of the media, methods orcompositions detailed herein.

TABLE 3 Exemplary reference standard values Reference standard Referencestandard value (a) Brown from about B1 to about B9; from about B1 toabout B8; from about B1 to about B7; from about B1 to about B6; fromabout B1 to about B5; from about B1 to about B4; from about B1 to aboutB3; from about B1 to about B2; from about B2 to about B9; from about B3to about B9; from about B4 to about B9; from about B5 to about B9; fromabout B6 to about B9; from about B7 to about B9; from about B8 to aboutB9; from about B2 to about B8; from about B3 to about B7; from about B4to about B6; from about B5 to about B7; from about B6 to about B8; aboutany of B1 or B2 or B3 or B4 or B5 or B6 or B7 or B8 or B9; at leastabout any of B1 or B2 or B3 or B4 or B5 or B6 or B7 or B8 or B9.Preferably B3 to B9. Most preferably B4 to B9. (b) Brownish- from aboutBY1 to about BY7; from about BY1 to about BY6; from Yellow about BY1 toabout BY5; from about BY1 to about BY4; from about BY1 to about BY3;from about BY1 to about BY2; from about BY2 to about BY7; from about BY3to about BY7; from about BY4 to about BY7; from about BY5 to about BY7;from about BY6 to about BY7; from about BY2 to about BY6; from about BY3to about BY5; from about BY4 to about BY6; from about BY5 to about BY6;about any of BY1 or BY2 or BY3 or BY4 or BY5 or BY6 or BY7; at leastabout any of BY1 or BY2 or BY3 or BY4 or BY5 or BY6 or BY7. PreferablyBY3 to BY7. Most preferably BY4 to BY7. (c) Yellow from about Y1 toabout Y7; from about Y1 to about Y6; from about Y1 to about Y5; fromabout Y1 to about Y4; from about Y1 to about Y3; from about Y1 to aboutY2; from about Y2 to about Y7; from about Y3 to about Y7; from about Y4to about Y7; from about Y5 to about Y7; from about Y6 to about Y7; fromabout Y2 to about Y6; from about Y3 to about Y5; from about Y4 to aboutY6; from about Y5 to about Y6; about any of Y1 or Y2 or Y3 or Y4 or Y5or Y6 or Y7; at least about any of Y1 or Y2 or Y3 or Y4 or Y5 or Y6 orY7. Preferably Y3 to Y7. Most preferably Y4 to Y7. (d) Greenish- fromabout GY1 to about GY7; from about GY1 to about GY6; from Yellow aboutGY1 to about GY5; from about GY1 to about GY4; from about GY1 to aboutGY3; from about GY1 to about GY2; from about GY2 to about GY7; fromabout GY3 to about GY7; from about GY4 to about GY7; from about GY5 toabout GY7; from about GY6 to about GY7; from about GY2 to about GY6;from about GY3 to about GY5; from about GY4 to about GY6; from about GY5to about GY6; about any of GY1 or GY2 or GY3 or GY4 or GY5 or GY6 orGY7; at least about any of GY1 or GY2 or GY3 or GY4 or GY5 or GY6 orGY7. Preferably GY3 to GY7. Most preferably GY4 to GY7. (e) Red fromabout R1 to about R7; from about R1 to about R6; from about R1 to aboutR5; from about R1 to about R4; from about R1 to about R3; from about R1to about R2; from about R2 to about R7; from about R3 to about R7; fromabout R4 to about R7; from about R5 to about R7; from about R6 to aboutR7; from about R2 to about R6; from about R3 to about R5; from about R4to about R6; from about R5 to about R6; about any of R1 or R2 or R3 orR4 or R5 or R6 or R7; at least about any of R1 or R2 or R3 or R4 or R5or R6 or R7. Preferably R3 to R7. Most preferably R4 to R7.

In another example, the polypeptides produced by the methods detailedherein with the media described herein (or present in the compositionsprovided) may be assessed for color with a quantitative assay. In someembodiments, the quantitative assay can be done using an automatedprocess. In some embodiments, a higher value (e.g., higher numericalvalue) provided by the quantitative assay indicates a higher colorintensity and a lower value (e.g., lower numerical value) indicates alower color intensity.

A color assay detailed herein may find use in assessing color of anysolution (e.g., a polypeptide-containing solution), including, but notlimited to, the polypeptide compositions provided herein.

IV. Compositions and Pharmaceutical Formulations

Compositions comprising the cell culture medium and one or more othercomponent, such as a cell or a desired polypeptide (e.g., an antibody),are also provided. A cell comprising a nucleic acid encoding apolypeptide of interest (e.g., an antibody) can secrete the polypeptideinto a cell culture medium of the invention during cell culture.Accordingly, compositions of the invention can comprise a cell thatproduces the polypeptide and a cell culture medium provided herein thatthe polypeptide is secreted into. Compositions comprising the producedpolypeptide and a cell culture medium provided herein are alsocontemplated. In some aspects of the invention, a composition comprises(a) a cell comprising a nucleic acid encoding a polypeptide; and (b) acell culture medium are provided herein. In some aspects, thecomposition comprises (a) a polypeptide; and (b) a cell culture mediumas provided herein, wherein the polypeptide is secreted into the mediumby a cell comprising an isolated nucleic acid encoding the polypeptide.In other aspects, the composition comprises: (a) a polypeptide; and (b)a cell culture medium as provided herein, wherein the polypeptide isreleased into the medium by lysis of a cell comprising an isolatednucleic acid encoding the polypeptide. The cell of the composition maybe any cell detailed herein (e.g., a CHO cell) and the medium of thecomposition may be any medium detailed herein, such as a mediumcomprising one or more compounds as detailed in Table 1 or Table 2.Likewise, the polypeptide of the composition may be any polypeptidedetailed herein, such as an antibody. In some aspects, the compositionmay have a color. In some embodiments, the color is determined,measured, or assessed by use of one or more visual color standards. Thevisual color standard can be an international or national color standardsuch as, but not limited to, the United States Pharmacopoeia colorstandard and the European Pharmacopoeia color standard. See USP-24Monograph 631 Color and Achromaticity. United States Pharmacopoeia Inc.,2000, p. 1926-1927 and Council of Europe. European Pharmacopoeia, 2008,7^(th) Ed. P. 22. Accordingly, in some embodiments, a compositioncomprising (a) a polypeptide; and (b) a cell culture medium providedherein is assessed for color intensity. In a further embodiment, thepolypeptide is isolated and/or purified before assessment of colorintensity. In some embodiments, a color intensity of a compositioncomprising (a) a polypeptide; and (b) a cell culture medium providedherein is used to predict the color intensity of the final proteincomposition. For example, a composition comprising a polypeptide and acell culture medium provided herein is measured for color intensityusing the COC assay as described herein. If the color intensity value isgreater than B3, B4, B5, B6, B7, B8, or B9 then there is an increasedlikelihood that the final protein composition will have a colorintensity value of greater than B3, B4, B5, B6, B7, B8, or B9. In someembodiments, the composition comprising a polypeptide and the cellculture medium is subjected to at least one purification step beforemeasurement of color intensity. In some embodiments, the final proteincomposition is a pharmaceutical formulation. In some aspects, acomposition as provided herein comprises a polypeptide at aconcentration of at least about 1 mg/mL, 10 mg/mL or 25 mg/mL or 50mg/mL or 75 mg/mL to about 100 mg/mL or at a concentration of about 1mg/mL, 10 mg/mL or 25 mg/mL or 50 mg/mL or 75 mg/mL to about 100 mg/mL.In some aspects, a composition as provided herein comprises apolypeptide at a concentration of at least 100 mg/mL or 125 mg/mL or 150mg/mL or at a concentration of about 100 mg/mL or 125 mg/mL or 150 mg/mLor 175 mg/mL or 200 mg/mL.

Compositions (e.g., pharmaceutical formulations) of the polypeptides(e.g. a therapeutic polypeptide) produced by any of the methodsdescribed herein are prepared by mixing a polypeptide having the desireddegree of purity with one or more optional pharmaceutically acceptablecarriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.(1980)), in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers, antioxidants, preservatives, low molecularweight (less than about 10 residues) polypeptides, proteins; hydrophilicpolymers; amino acids; monosaccharides, disaccharides, and othercarbohydrates, chelating agents, sugars, salt-forming counter-ions,metal complexes (e.g. Zn-protein complexes), and/or non-ionicsurfactants. Exemplary lyophilized polypeptide formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous polypeptide formulationsinclude those described in U.S. Pat. No. 6,171,586 and WO2006/044908,the latter formulations including a histidine-acetate buffer. In someembodiments, the pharmaceutical formulation is administered to a mammalsuch as a human. Pharmaceutical formulations of the polypeptide (e.g.,an antibody) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Accordingly,polypeptide-containing formulations as provided herein may be suitablefor injection, such as subcutaneous injection into an individual (e.g.,subcutaneous injection into a human). The pharmaceutical formulations tobe used for in vivo administration are generally sterile. Sterility maybe readily accomplished, for example by filtration through sterilefiltration membranes.

In some aspects, a composition (e.g., pharmaceutical formulation) asprovided herein comprises a polypeptide (e.g., a therapeuticpolypeptide) at a concentration of at least about 1 mg/mL, 10 mg/mL, 25mg/mL, 50 mg/mL, or 75 mg/mL, or at a concentration of about 1 mg/mL,about 10 mg/mL, about 25 mg/mL, about 50 mg/mL, or about 75 mg/mL up toabout 100 mg/mL. In other aspects, a composition (e.g., pharmaceuticalformulation) as provided herein comprises a polypeptide (e.g., atherapeutic polypeptide) at a concentration of at least about 100 mg/mL,125 mg/mL, 150 mg/mL, 200 mg/mL, or 250 mg/mL, or at a concentration ofabout 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL,about 200 mg/mL, or about 250 mg/mL. In some embodiments, apharmaceutical formulation as provided herein comprises a polypeptide ata concentration greater than at least about 1 mg/mL, at least about 10mg/mL, at least about 25 mg/mL, at least about 50 mg/mL, or at leastabout 75 mg/mL and has a color intensity value greater than B3, B4, B5,B6, B7, B8, or B9 as measured by the COC assay. In some embodiments, apharmaceutical formulation as provided herein comprises a polypeptide ata concentration greater than at least about 100 mg/mL, at least about125 mg/mL, at least about 150 mg/mL, or at least about 200 mg/mL and hasa color intensity value greater than B3, B4, B5, B6, B7, B8, or B9 asmeasured by the COC assay. In some aspects, the color intensity value asdetermined by the COC assay can be any one of, but not limited to, B,BY, Y, GY, or R, wherein higher values indicate a lighter colorintensity. In some aspects, a pharmaceutical formulation as providedherein comprises a polypeptide at a concentration greater than at leastabout 1 mg/mL, at least about 10 mg/mL, at least about 25 mg/mL, atleast about 50 mg/mL, or at least about 75 mg/mL and has a colorintensity value less than a color intensity value of a referencesolution as measured by a color assay. In some aspects, a pharmaceuticalformulation as provided herein comprises a polypeptide at aconcentration greater than at least about 100 mg/mL, at least about 125mg/mL, at least about 150 mg/mL, or at least about 200 mg/mL and has acolor intensity value less than a color intensity value of a referencesolution as measured by a color assay. For example, the color intensityof a composition (e.g., pharmaceutical formulation) comprising apolypeptide (e.g., a therapeutic polypeptide) can be reduced by at least0.1% or by about 5% to about 50% as compared to a composition comprisingthe polypeptide produced by a cell cultured in a cell culture mediumthat does not comprise the one or more of components of Table 1 or Table2.

V. Articles of Manufacture or Kits

A kit for supplementing a cell culture medium with chemically definedconstituents is described. The kit may contain dried constituents to bereconstituted, and may also contain instructions for use (e.g., for usein supplementing a medium with the kit constituents). The kit maycontain the constituents provided herein in amounts suitable tosupplement a cell culture medium. In some aspects, the kit contains oneor more constituent selected from the group consisting of hypotaurine,s-carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine,lipoic acid, and quercitrin hydrate in amounts to supplement a cellculture medium with a constituent concentration as provided in Table 1or Table 2. In some embodiments, a kit comprises one or more of: (a)hypotaurine in an amount to provide from about 2.0 mM to about 50.0 mMhypotaurine in the cell culture medium; (b) s-carboxymethylcysteine inan amount to provide from about 8.0 mM to about 12.0 mMs-carboxymethylcysteine in the cell culture medium; (c) carnosine in anamount to provide from about 8.0 mM to about 12.0 mM carnosine in thecell culture medium; (d) anserine in an amount to provide from about 3.0mM to about 5.0 mM anserine in the cell culture medium; (e) butylatedhydroxyanisole in an amount to provide from about 0.025 mM to about0.040 mM butylated hydroxyanisole; (f) lipoic acid in an amount toprovide from about 0.040 mM to about 0.060 mM lipoic acid in the cellculture medium; and (g) quercitrin hydrate in an amount to provide fromabout 0.010 mM to about 0.020 mM quercitrin hydrate in the cell culturemedium. In some aspects, the kit contains one or more constituent,wherein the one or more constituent is hypotaurine or an analog orprecursor thereof. In some embodiments, the hypotaurine or an analog orprecursor thereof is selected from the group consisting of hypotaurine,s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, andtaurine. In some embodiments, a kit for supplementing a cell culturemedium with chemically defined constituents, the kit comprisinghypotaurine or an analog or precursor thereof at a concentration of atleast about 0.0001 mM, and wherein the hypotaurine or an analog orprecursor is selected from the group consisting of hypotaurine,s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, andtaurine.

In another aspect of the invention, an article of manufacture isprovided comprising a container which holds the cell culture medium ofthe invention and optionally provides instructions for its use. Suitablecontainers include, for example, bottles and bags. The container may beformed from a variety of materials such as glass or plastic. Thecontainer holds the cell culture medium and the label on, or associatedwith, the container may indicate directions for use (e.g., for use inculturing cells). The article of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents and package inserts with instructions for use.

The following Examples are provided to illustrate but not to limit theinvention.

EXAMPLES

Media have been identified that produce a protein product (e.g., aprotein drug product) with acceptable quality attributes, such asreduced color intensity, particularly when the protein product ispresent as a concentrated solution (e.g., to a concentration of at leastabout 1 mg/mL or at least about 100 mg/mL). Methods of culturing cellsin the media provided herein are described, as are methods of producinga polypeptide using the media. A media may in one aspect comprisehypotaurine. In some of the aspects provided herein, the media comprisesone or more hypotaurine analog or precursor thereof, such ascarboxymethylcysteine. Each of the media constituents may be present inany value provided throughout. The media may be chemically defined orchemically undefined. The media may reduce the presence of reactiveoxygen species when used in a method of polypeptide production ascompared to the polypeptide produced in different media. The media findsuse through all phases of cell culture and polypeptide production andmay be used in the basal and/or feed medium. A polypeptide produced byany of the methods described herein is provided, as is a pharmaceuticalcomposition comprising a polypeptide produced as detailed herein. In oneaspect, the pharmaceutical compositions comprise the polypeptide at aconcentration of at least or about any of 100 mg/mL, 125 mg/mL, or 150mg/mL. Methods of making and compositions comprising antibodies areparticularly contemplated. Kits for supplementing a cell culture mediumwith chemically defined constituents are also described.

Example 1: Identification of Antioxidant Compounds Capable of ReducingColor in Antibody Compositions

Compounds that have been reported to react with an oxidant were screenedfor their ability to reduce the color of protein containing compositions(Table 4). For antioxidant screening, a total volume of 40 ml media wasprepared by mixing 1 part basal Media 1 and 0.3 part feed Media 2 tominor a representative ratio of media used in cell culture conditions(Table 5). The mixture of Media 1 and Media 2, which was previouslyshown to increase the color intensity of antibody-containing solutionswhen used for culturing antibody-producing cells, was supplemented withone of 30 antioxidant compounds and spiked with 2 g/L IgG1 monoclonalantibody. The samples were incubated at 37° C. with shaking at 250 rpmfor a five day incubation period. Two control samples were included inthe screening assay: 1) a 40 ml sample of a Media 1 and Media 2 mixturecontaining 2 g/L IgG1 monoclonal antibody that was incubated for 5 daysat 37° C. with shaking at 250 rpm without antioxidant (positivecontrol), and 2) a 40 mL sample of a media mixture prepared by mixing 1part basal Media 3 and 0.3 part feed Media 4 (Table 5), which waspreviously shown to reduce the color intensity of antibody-containingsolutions when used for culturing antibody-producing cells, spiked with2 g/L IgG1 monoclonal antibody and incubated for 5 days at 37° C. withshaking at 250 rpm without antioxidant (negative control).

TABLE 4 Representative compounds screened for reduction of color 1X TestAntioxidant IUPAC CAS # Concentration 2,3-tert-butyl-4-2-tert-butyl-4-methoxyphenol 25013-16-5 34.68 μM hydroxyanisole2,6-di-tert-butyl-4- 2,6-di-tert-butyl-4-methylphenol 97123-41-6 102.11μM methylphenol 3-aminopropane-1- 3-aminopropane-1-sulfonic acid3687-18-1 9.16 mM sulfonic acid AdenosylhomocysteineS-(5′-Deoxyadenos-5′-yl)-L-homocysteine 979-92-0 10.41 μM Anserine(2S)-2-(3-aminopropanamido)-3-(1-methyl-1H- 10030-52-1 4.12 mMimidazol-5-yl)propanoic acid; nitric acid B-Alanine 3-aminopropanoicacid 107-95-9 9.16 mM B-carotene 1,3,3-trimethyl-2- 7235-40-7 9.31 μM[(1E,3E,5E,7E,9E,11E,13E,15E,17E)-3,7,12,16-tetramethyl-18-(2,6,6-trimethylcyclohex-1-en-1-yl)octadeca-1,3,5,7,9,11,13,15,17-nonaen-1- yl]cyclohex-1-ene Butylated2-tert-butyl-4-methoxyphenol 25013-16-5 31.62 μM hydroxyanisoleButylated 2,6-di-tert-butyl-4-methylphenol 128-37-0 124.80 μMhydroxytoluene Carnosine (2S)-2-(3-aminopropanamido)-3-(1H-imidazol-5-305-84-0 10.00 mM yl)propanoic acid Carvedilol[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl][2-(2- 72956-09-3 21.53 μMmethoxyphenoxy)ethyl]amine Curcumin(1E,4Z,6E)-5-hydroxy-1,7-bis(4-hydroxy-3- 458-37-7 49.95 μMmethoxyphenyl)hepta-1,4,6-trien-3-one Cysteamine 2-aminoethane-1-thiol60-23-1 12.00 mM Cysteamine hydrogen 2-aminoethane-1-thiol chloride156-57-0 10.00 mM hydrochloride Dexamethasone(1R,2S,10S,11S,13R,14R,15S,17S)-1-fluoro- 50-02-2 9.56 μM14,17-dihydroxy-14-(2-hydroxyacetyl)-2,13,15-trimethyltetracyclo[8.7.0.0{circumflex over ( )}{2,7}.0{circumflex over( )}{11,15}]hepta deca-3,6-dien-5-one Diallyldisulfide3-(prop-2-en-1-ylsulfanyl)prop-1-ene 592-88-1 1.00 mM DL-Lanthionine2-amino-3-[(2-amino-2- 3183-08-2 97.96 μMcarboxyethyl)sulfanyl]propanoic acid DL-Thiorphan2-(2-benzyl-3-sulfanylpropanamido)acetic acid 76721-89-6 0.10 mMEthoxyquin 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline 91-53-2 49.99μM Gallic acid 3,4,5-trihydroxybenzoic acid 149-91-7 14.11 μM Gentisicacid sodium sodium 2,5-dihydroxybenzoate 4955-90-2 2.84 mM salt hydrateGlutathione 2-amino-4-({1-[(carboxymethyl)carbamoyl]-2- 70-18-8 2.0 mMsulfanylethyl}carbamoyl)butanoic acid Glutathione disulfide2-amino-4-[(2-{[2-(4-amino-4- 27025-41-8 2.0 mM carboxybutanamido)-2-[(carboxymethyl)carbamoyl]ethyl]disulfanyl}-1-[(carboxymethyl)carbamoyl]ethyl)carbamoyl]butanoic acid Glutathionereduced (2S)-2-amino-4-{[(1R)-1- 92614-59-0 0.93 mM ethyl ester[(carboxymethyl)carbamoyl]-2- sulfanylbutyl]carbamoyl}butanoic acidGlycine 2-aminoacetic acid 56-40-6 13.32 mM Hydrocortisone(1S,2R,10S,11S,14R,15S,17S)-14,17-dihydroxy- 50-23-7 55.03 mM14-(2-hydroxyacetyl)-2,15- dimethyltetracyclo[8.7.0.0{circumflex over( )}{2,7}.0{circumflex over ( )}{11,15}]hepta dec-6-en-5-one Hypotaurine2-aminoethane-1-sulfinate 300-84-5 9.16 mM Isethionic acid ammonium2-hydroxyethane-1-sulfonate 57267-78-4 9.16 mM ammonium saltL-Cysteine-glutathione (2S)-2-amino-4-{[(1R)-2-{[(2R)-2-amino-3,3-13081-14-6 0.73 mM Disulfide dihydroxypropyl]sulfanyl}-1-[(carboxymethyl)carbamoyl]-2- sulfanylideneethyl]carbamoyl}butanoic acidL-Cysteinesulfinic acid (2R)-2-amino-3-[(R)-sulfino]propanoic acid207121-48-0 9.15 mM monohydrate hydrate Lipoic Acid5-[(3R)-1,2-dithiolan-3-yl]pentanoic acid 1200-22-2 50.40 μM Lipoic acidreduced 6,8-disulfanyloctanoic acid 462-20-4 48.00 μM Mercaptopropionyl2-(2-sulfanylpropanamido)acetic acid 1953-02-2 10.00 mM glycineMethionine 2-amino-4-(methylsulfanyl)butanoic acid 59-51-8 5.00 mMMethylenebis(3- 3-({[(2- 4265-57-0 0.99 mM thiopropionic acid)carboxyethyl)sulfanyl]methyl}sulfanyl)propanoic acid Oxalic acid oxalicacid 144-62-7 500.94 μM Quercetrin hydrate2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3- 522-12-3 13.94 μM{[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}-4H-chromen-4-one Resveratrol5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3- 501-36-0 98.58 μM diolRetinoic acid (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6- 302-79-4 2.0 μMtrimethylcyclohex-1-en-1-yl)nona-2,4,6,8- tetraenoic acidS-Carboxymethyl-L- (2R)-2-amino-3- 638-23-3 10.00 mM cysteine[(carboxymethyl)sulfanyl]propanoic acid Selenium selanylidene 7782-49-21.40 μM Selenomethionine (2S)-2-amino-4-(methylselanyl)butanoic acid3211-76-5 30.09 μM Silver silver(1+) ion (diethylcarbamothioyl)sulfanide1470-61-7 0.10 mM diethyldithiocarbamate Taurine2-aminoethane-1-sulfonic acid 107-35-7 5.00 mM Thiolactic acid2-sulfanylpropanoic acid amine 79-42-5 10.00 mM Tricine2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2- 5704-04-1 4.46 mMyl]amino}acetic acid Vitamin C 2-(1,2-dihydroxyethyl)-4,5-dihydroxy-2,3-50-81-7 9.82 μM dihydrofuran-3-one Vitamin E(2R)-2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12- 10191-41-0 27.86 μMtrimethyltridecyl]-3,4-dihydro-2H-1-benzopyran- 6-ol 1X testconcentration indicates final concentration in the media

After incubation, the monoclonal antibody was purified using protein Aaffinity chromatography. The pool was concentrated to 150 g/L usingAmicon Centricon centrifugal filter devices (Millipore Corporation,Billerica, Mass.). Color intensity of the concentrated antibodycomposition was measured in the concentrated protein A pool using anassay wherein higher numerical values indicate higher color intensityand lower numerical values indicate lower color intensity. The numericalresults were normalized to the positive control, where the value for thepositive control was set at 0% change in color intensity. Of the 30antioxidant compounds tested several compounds, such as gentisic acid,cysteamine, hydrocortisone, and mercaptopropionyl glycine, were found toincrease the color of the antibody composition (FIG. 1). In comparison,six of the compounds such as hypotaurine, anserine, butylatedhydroxyanisole, carno sine, lipoic acid, and quercitrin hydrate, werefound to reduce the color of the antibody composition (FIG. 2). Of theantioxidants that reduced color intensity, hypotaurine demonstrated thegreatest effect by reducing the color intensity of theantibody-containing compositions by approximately 25%. Taurine, ananalog of hypotaurine, also reduced color intensity by approximately 5%.

TABLE 5 Representative components in media compositions tested Media 1Media 2 Media 3 Media 4 Media Components (Basal) (Feed) (Basal) (Feed)Iron (μM) 75^(a )  0 18^(b )  0 Vitamin B2 (mg/L) 1.41 10 0.25 0 VitaminB6/Pyridoxine 15.42  7 5.35 0 (mg/L) Vitamin B6/Pyridoxal (mg/L) 0   600   0 Vitamin B9 (mg/L) 9.93 197 8.61 0 Vitamin B12 (mg/L) 3.05 48 1.760 Cysteine (mg/L) 525    1500 0   1500 Cystine (mg/L) 0   0 480    0Hydrocortisone (nM) 150    0 150    0 ^(a)Iron source is ferrous sulfate^(b)Iron source is ferric citrate

Example 2: Characterization of Antioxidant Compounds Capable of ReducingColor Intensity in Antibody Compositions Isolated fromAntibody-Producing Cell Lines

The ability of hypotaurine to reduce color intensity in antibodycontaining compositions obtained directly from cell cultures wasevaluated. For these studies a shaker flask cell culture model wasutilized that was found to be representative of larger scale 2 L cellculture. Briefly, for the shaker flask cell culture model, antibodyproducing CHO cells were inoculated at approximately 1.0×10⁶ cells/mL ina 250 mL flask containing 100 mL of basal Media 1 or basal Media 3. Forthe larger scale 2 L cell cultures, antibody producing CHO cells wereinoculated at approximately 1.0×10⁶ cells/mL in 2-liter stirredbioreactors (Applikon, Foster City, Calif.) containing 1 L of basalMedia 1 or basal Media 3. For the larger scale cell growth model, cellswere cultured in fed-batch mode with addition of 100 mL of feed Media 2if cultured in basal Media 1, or with 100 mL of feed Media 4 if culturedin basal Media 3, per liter of cell culture fluid at days 3, 6 and 9 forinitiation of the production phase. For the shaker flask cell culturemodel, the cells were cultured in fed-batch mode with addition of 10 mLof feed Media 2 if cultured in basal Media 1, or with 10 mL of feedMedia 4 if cultured in basal Media 3, per liter of cell culture fluid atdays 3, 6 and 9 for initiation of the production phase. Theconcentration of glucose was analyzed every day and if the glucoseconcentration fell below 3 g/L, it was replenished from a 500 g/L stocksolution of glucose for prevention of glucose depletion. Reactors wereequipped with calibrated dissolved oxygen, pH and temperature probes.Dissolved oxygen was controlled on-line through sparging with air and/oroxygen. For the larger scale 2 L cell culture, pH was controlled throughaddition of CO₂ or Na₂CO₃ and antifoam was added to the cultures asneeded. The cell cultures were maintained at pH 7.0 and a temperature of37° C. from days 0 through 3, and then at 35° C. after day 3. The cellcultures were agitated at 275 rpm and the dissolved oxygen level was at30% of air saturation. For the shaker flask cell cultures, cultures wereplaced on a shaker platform and agitated at 150 rpm in a 5% CO₂incubator with a temperature of 37° C. from day 0 up to day 3 of thecell culture cycle with a temperature shift to 35° C. on day 4 until theend of the cell culture cycle at day 14. Osmolality was monitored usingan osmometer from Advanced Instruments (Norwood, Mass.). Offline pH andmetabolite concentrations were also determined daily using a NovaBioprofile 400 (Nova Biomedical, Waltham, Mass.). Viable cell density(VCC) and cell viability was measured daily using a ViCell® automatedcell counter (Beckman Coulter, Fullerton, Calif.). The cell culturefluid was collected daily by centrifuging 1 mL of cell culture fluid fordetermination of antibody titer using high performance liquidchromatography. At the end of the cell culture duration on day 14, thecell culture fluid from all samples was harvested by centrifugation. Themonoclonal antibody in the harvested cell culture fluid was purifiedusing protein A affinity chromatography. The protein A pool wasconcentrated to 150 g/L using Amicon Centricon centrifugal filterdevices (Millipore Corporation, Billerica, Mass.). Color intensity ofthe concentrated antibody composition was measured in the concentratedprotein A pool using an assay wherein higher numerical values indicatehigher color intensity and lower numerical values indicate lower colorintensity. Growth as measured by VCC (FIG. 3A) and cell viability (FIG.3B) were comparable between the larger scale (2 L) and shaker flask (SF)cell culture models regardless of the media used. Antibody productionwas slightly lower in the shaker flask cell culture model with thehighest antibody production observed in the larger scale cell culturemodel incubated in Media 1 and Media 2 (FIG. 3C). Color intensity ofantibody compositions obtained from the shaker flask cell culture modelwas lower at a value of 1.07 when cultured in Media 3 and Media 4 ascompared to antibody compositions obtained from shaker flask cellculture compositions when cultured in Media 1 and Media 2 which had avalue of 2.25. These experiments established that the shaker flask modelwas comparable to the 2 L cell culture model and was suitable for use insubsequent experiments.

For experimentation with cell culture media compositions that weresupplemented with the antioxidant hypotaurine, antibody producing CHOcells were inoculated at approximately 1.0×10⁶ cells/mL in a 250 mLflask containing 100 mL of basal Media 1. Media 1 was supplemented with9.16 mM (100%), 4.58 mM (50%), or 2.29 mM (25%) hypotaurine for use incell culture on Day 0. The cells were cultured in fed-batch mode withaddition of 10 mL of feed Media 2 per liter of cell culture fluid atDays 3, 6 and 9 for initiation of the production phase. An additionalexperimental sample involved the incremental addition of 9.16 mMhypotaurine over the cell culture period. Specifically, 2.29 mM (25%)hypotaurine was added on Day 0 of cell culture in basal Media 1, and 25%was added on Day 3, Day 6 and Day 9 in feed Media 2. A positive controlwas included by culturing cells in Media 1 and 2 without hypotaurinesupplementation. The negative control was included by culturing cellscultured in Media 3 and Media 4 without hypotaurine supplementation. Asdescribed above, the concentration of glucose was analyzed every day andif the glucose concentration fell below 3 g/L, it was replenished from a500 g/L stock solution of glucose for prevention of glucose depletion.The cell cultures were maintained at pH 7.0 and a temperature of 37° C.from days 0 through 3, and then at 35° C. after day 3. The cell cultureswere agitated at 275 rpm and the dissolved oxygen level was at 30% ofair saturation. VCC and cell viability was measured daily using aViCell® automated cell counter (Beckman Coulter, Fullerton, Calif.). Thecell culture fluid was collected daily by centrifuging 1 mL of cellculture fluid for determination of antibody titer using high performanceliquid chromatography. At the end of the cell culture duration on day14, the cell culture fluid from all samples was harvested bycentrifugation. The monoclonal antibody in the harvested cell culturefluid was purified using protein A affinity chromatography. The proteinA pool was concentrated to 150 g/L using Amicon Centricon centrifugalfilter devices (Millipore Corporation, Billerica, Mass.). Colorintensity of the concentrated antibody composition was measured in theconcentrated protein A pool using an assay wherein higher numericalvalues indicate higher color intensity and lower numerical valuesindicate lower color intensity. The numerical results were normalized tothe positive control, where the value for the positive control was setat 0% change in color intensity. Growth as measured by VCC (FIG. 4A) andcell viability (FIG. 4B) was comparable among all the cell culturestested. Furthermore, with the exception of incremental addition ofhypotaurine, cell cultures cultured in media supplemented withhypotaurine produced the same level of antibody titers as cell culturescultured in media not containing hypotaurine (FIG. 4C). Color intensitywas found to be reduced with higher concentration of hypotaurine withthe greatest reduction observed in media containing 9.16 mM hypotaurine(FIG. 5). This reduction in color intensity was optimal when hypotaurinewas added as a bolus at Day 1 rather than added incrementally over thecourse of cell culture incubation. Comparison of color intensity valuesobtained from cell culture experiments and incubation experiments (SeeExample 1) demonstrated that the results of the incubation screeningexperiments (FIG. 5, empty circles) correlated well with the resultsfrom cell culture experiments (FIG. 5, filled circles).

Similar experiments were conducted for antibody compositions isolatedfrom cell cultures harvested in basal Media 3 and feed Media 4 todetermine if the color reducing effect of hypotaurine extended to othercell culture media. Briefly, as above, antibody producing CHO cells wereinoculated at approximately 1.0×10⁶ cells/mL in a 250 mL flaskcontaining 100 mL of basal Media 3. Media 3 was supplemented with 12.95mM (1×), 25.9 mM (2×), or 38.85 mM (3×) hypotaurine for use in cellculture on Day 0. The cells were cultured in fed-batch mode withaddition of 10 mL of feed Media 4 per liter of cell culture fluid atDays 3, 6 and 9 for initiation of the production phase. A positivecontrol was included by culturing cells in Media 1 and 2 withouthypotaurine supplementation. The cultures were placed on a shakerplatform and agitated at 150 rpm in a 5% CO₂ incubator with atemperature of 37° C. from day 0 up to day 3 of the cell culture cyclewith a temperature shift to 35° C. on day 4 until the end of the cellculture cycle at day 14. Osmolality, offline pH and metaboliteconcentrations were measured as described above. VCC and cell viabilitywas measured daily using a ViCell® automated cell counter (BeckmanCoulter, Fullerton, Calif.). The cell culture fluid was collected dailyby centrifuging 1 mL of cell culture fluid for determination of antibodytiter using high performance liquid chromatography. At the end of thecell culture duration on day 14, the cell culture fluid from all sampleswas harvested by centrifugation. The monoclonal antibody in theharvested cell culture fluid was purified using protein A affinitychromatography. The protein A pool was concentrated to 150 g/L usingAmicon Centricon centrifugal filter devices (Millipore Corporation,Billerica, Mass.). Color intensity of the concentrated antibodycomposition was measured in the concentrated protein A pool using anassay wherein higher numerical values indicate higher color intensityand lower numerical values indicate lower color intensity. The numericalresults were normalized to the positive control, where the value for thepositive control was set at 0% change in color intensity. Colorintensity was found to be reduced with higher concentration ofhypotaurine with the greatest reduction observed in media containing38.85 mM hypotaurine (FIG. 6).

Example 3: Characterization of Hypotaurine Analogs in Reduction of Colorin Antibody Compositions Isolated from Antibody-Producing Cell Lines

Hypotaurine analogs were tested to assess if they demonstrated a colorreducing effect in antibody containing compositions. Antibody producingCHO cells were inoculated at approximately 1.0×10⁶ cells/mL in 2-literstirred bioreactors (Applikon, Foster City, Calif.) containing 1 L ofbasal Media 1 supplemented with 12.95 mM hypotaurine or 10 mMcarboxymethylcysteine (CAS number 638-23-3). Cells were cultured infed-batch mode with addition of 100 mL of feed Media 2 per liter of cellculture fluid at days 3, 6 and 9 for initiation of the production phase.A positive control was included by culturing cells in Media 1 and 2without hypotaurine supplementation. The concentration of glucose wasanalyzed every day and if the glucose concentration fell below 2 g/L, itwas replenished from a 1.5 g/L stock solution of glucose for preventionof glucose depletion. Reactors were equipped with calibrated dissolvedoxygen, pH and temperature probes. Dissolved oxygen was controlledon-line through sparging with air and/or oxygen. pH was controlledthrough addition of CO₂ or Na₂CO₃ and antifoam was added to the culturesas needed. The cell cultures were maintained at pH 7.0 and a temperatureof 37° C. from days 0 through 3, and then at 35° C. after day 3. Thecell cultures were agitated at 275 rpm and the dissolved oxygen levelwas at 30% of air saturation. Osmolality was monitored using anosmometer from Advanced Instruments (Norwood, Mass.). Offline pH andmetabolite concentrations were also determined daily using a NovaBioprofile 400 (Nova Biomedical, Waltham, Mass.). VCC and cell viabilitywas measured daily using a ViCell® automated cell counter (BeckmanCoulter, Fullerton, Calif.). The cell culture fluid was collected dailyby centrifuging 1 mL of cell culture fluid for determination of antibodytiter using high performance liquid chromatography. At the end of thecell culture duration on day 14, when the amount of protein in theculture was approximately 2-10 g/L, the cell culture fluid from allsamples was harvested by centrifugation. The monoclonal antibody in theharvested cell culture fluid was purified using protein A affinitychromatography. The protein A pool was concentrated to 150 g/L usingAmicon Centricon centrifugal filter devices (Millipore Corporation,Billerica, Mass.). Color intensity of the concentrated antibodycomposition was measured in the concentrated protein A pool using twodifferent assays wherein higher numerical values indicated higher colorintensity and lower numerical values indicated lower color intensity.Growth as measured by VCC (FIG. 7A) and cell viability (FIG. 7B) wascomparable among all cell cultures tested. Cell cultures cultured inmedia supplemented with hypotaurine or carboxymethylcysteine producedcomparable levels of antibody titers (FIG. 8). Using a specific colorassay, color intensity of isolated antibody composition was found to bereduced by 27% and 13% when antibody-producing cells were cultured mediasupplemented with hypotaurine and carboxymethylcysteine, respectively(FIG. 9A). This color intensity reduction was confirmed by using asecond color assay which detected an approximate 17% and 13% colorintensity reduction in antibody compositions isolated from cell culturedin media supplemented with hypotaurine and carboxymethylcysteine,respectively (FIG. 9B).

The invention claimed is:
 1. A method of producing a recombinantantibody composition with reduced color intensity, comprising the stepsof: culturing a cell comprising a nucleic acid encoding the recombinantantibody in a cell culture medium, wherein the cell culture mediumcomprises one or more of components (a)-(d): (a) hypotaurine, (b)s-carboxymethylcysteine, (c) butylated hydroxyanisole, and (d)quercitrin hydrate; and producing the recombinant antibody; wherein thecell culture medium comprising one or more of components (a)-(d) reducesthe color intensity of a composition comprising the recombinant antibodyproduced by the cell as compared to a composition comprising therecombinant antibody produced by the cell cultured in a cell culturemedium that does not comprise one or more of components (a)-(d).
 2. Themethod of claim 1, wherein the recombinant antibody is an antibodyfragment.
 3. The method of claim 1, wherein the recombinant antibody isan IgG1 antibody.
 4. The method of claim 1, 2 or 3, wherein therecombinant antibody is secreted into the cell culture medium.
 5. Themethod of claim 1, further comprising the step of recovering therecombinant antibody from the cell culture medium comprising one or moreof components (a)-(d).
 6. The method of claim 5, wherein a compositioncomprising the recovered recombinant antibody is a liquid composition ora non-liquid composition.
 7. The method of claim 6, wherein thecomposition comprising the recovered recombinant antibody appears as acolorless or slightly colored liquid.
 8. The method of claim 1, whereinthe cell culture medium comprising one or more of components (a)-(d)reduces the color intensity of a composition comprising the recombinantantibody produced by the cells by at least about 0.1% as compared to acomposition comprising the recombinant antibody produced by the cellcultured in a cell culture medium that does not comprise the one or moreof components (a)-(d).
 9. The method of claim 1, wherein the cellculture medium comprising one or more of components (a)-(d) reduces thecolor intensity of a composition comprising the recombinant antibodyproduced by the cells by about 5% to about 50% as compared to acomposition comprising the recombinant antibody produced by the cellcultured in a cell culture medium that does not comprise the one or moreof components (a)-(d).
 10. The method of claim 1, wherein the cellculture medium comprises the one or more components (a)-(d) in an amountselected from: (a) hypotaurine at a concentration from at least about0.0001 mM; (b) s-carboxymethylcysteine at a concentration from at leastabout 0.0001 mM; (c) butylated hydroxyanisole at a concentration from atleast about 0.0001 mM; and (d) quercitrin hydrate at a concentrationfrom at least about 0.0001 mM.
 11. The method of claim 10, wherein thecell culture medium comprises hypotaurine at a concentration from about2.0 mM to about 50.0 mM.
 12. The method of claim 10, wherein the cellculture medium comprises s-carboxymethylcysteine at a concentration fromabout 8.0 mM to about 12.0 mM.
 13. The method of claim 10, wherein thecell culture medium comprises butylated hydroxyanisole at aconcentration from about 0.025 mM to about 0.040 mM.
 14. The method ofclaim 10, wherein the cell culture medium comprises quercitrin hydrateat a concentration from about 0.010 mM to about 0.020 mM.
 15. The methodof claim 1, wherein the cell culture medium is a chemically defined cellculture medium.
 16. The method of claim 1, wherein the cell culturemedium is a chemically undefined cell culture medium.
 17. The method ofclaim 1, wherein the cell culture medium is a basal cell culture medium.18. The method of claim 1, wherein the cell culture medium is a feedcell culture medium.
 19. The method of claim 1, wherein the cell iscontacted with the cell culture medium during the cell's growth phase.20. The method of claim 1, wherein the cell is contacted with the cellculture medium during the cell's production phase.
 21. The method ofclaim 1, wherein the one or more of components (a)-(d) is added to thecell culture medium on at least one day of a cell culture cycle.
 22. Themethod of claim 1, wherein the one or more of components (a)-(d) isadded to the cell culture medium on day 0 of a 14 day cell culturecycle.
 23. The method of claim 1, wherein the cell is a mammalian cell.24. The method of claim 23, wherein the mammalian cell is a ChineseHamster Ovary (CHO) cell.